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The requirements of augmented signal contrast provided by nanoparticle tags in biosensor microscopy-based point-of-care technologies for cancer and infectious disease diagnostics can be addressed through metallo-dielectric nanoarchitectures that enhance optical scattering and absorption to provide digital resolution detection of single tags with simple instrumentation. Photonic Resonator Interferometric Scattering Microscopy (PRISM) enables label-free visualization of nanometer-scale analytes such as extracellular vesicles and virions, and its applicability can be extended to biomolecular analyte counting through nanoparticle tags. Here, we present template-free, linker-less cryosoret nano-assemblies fabricated via adiabatic cooling (−196 °C) as plasmonic nano-antennas that provide high scattering contrast in PRISM. Plasmonic Ag and Au nanomaterials and their cryosorets are evaluated through imaging experiments and simulations based on the finite element method to understand the photo-plasmonic coupling effect at the surface of a photonic crystal (PC) interface. The Ag and Au cryosorets provide at most 8.29-fold and 6.77-fold higher signal contrast compared to their singlet counterpart. Through the simulations, the averaged field magnitude enhancements of 2.77-fold and 3.68-fold are observed for Ag and Au cryosorets when interfacing with PCs compared to bare glass substrates. The hybrid coupling between the localized Mie and delocalized Bragg plasmons of cryosorets and the underlying PC's guided mode resonance provides insights for developing nano-assembly-based nano-tags for biosensing applications. [ABSTRACT FROM AUTHOR]

Diagnostic assays utilizing fluorescent reporters in the context of low abundance biomarkers for cancer and infectious disease can reach lower limits of detection through efficient collection of emitted photons into an optical sensor. In this work, we present the rational design, fabrication, and application of one-dimensional photonic crystal (PC) grating interfaces to accomplish a cost-effective prism-free, metal-free, and objective-free platform for augmentation of fluorescence emission collection efficiency. Guided mode resonance (GMR) of the PC is engineered to match the laser excitation (532 nm) and emission maximum (580 nm) of the radiating dipoles to arrive at optimized conditions. The photo-plasmonic hybrid nano-engineering using silver nanoparticles presented >110-fold steering fluorescence enhancement enabling placement of the sample between the excitation source and detector that are in a straight line. From the experimental and simulation inferences, we propose a radiating GMR model by scrutinizing the polarized emission properties of the hybrid substrate, in accordance with the radiating plasmon model. The augmented fluorescence intensity realized here with a simple detection instrument provides sub-nanomolar sensitivity to provide a path toward point-of-care scenarios. [ABSTRACT FROM AUTHOR]

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis‐ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of miRNAs using photonic resonator absorption microscopy. We achieve multiplex digital detection with sub‐attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. Compared with traditional qRT‐PCR, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31‐fold and 61‐fold enhancement in the limits of miRNA‐375 and miRNA‐21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low‐cost, and minimally invasive point‐of‐care testing. [ABSTRACT FROM AUTHOR]

Exosomal microRNAs (miRNAs) have considerable potential as pivotal biomarkers to monitor cancer development, dis‐ease progression, treatment effects and prognosis. Here, we report an efficient target recycling amplification process (TRAP) for the digital detection of miRNAs using photonic resonator absorption microscopy. We achieve multiplex digital detection with sub‐attomolar sensitivity in 20 minutes, robust selectivity for single nucleotide variants, and a broad dynamic range from 1 aM to 1 pM. Compared with traditional qRT‐PCR, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31‐fold and 61‐fold enhancement in the limits of miRNA‐375 and miRNA‐21 detection, respectively. The TRAP approach is ideal for exosomal or circulating miRNA biomarker quantification, where the miRNAs are present in low concentrations or sample volume, with potentials for frequent, low‐cost, and minimally invasive point‐of‐care testing. [ABSTRACT FROM AUTHOR]

Nanoscale fluorescence emitters are efficient for measuring biomolecular interactions, but their utility for applications requiring single-unit observations is constrained by the need for large numerical aperture objectives, fluorescence intermittency, and poor photon collection efficiency resulting from omnidirectional emission. Photonic crystal (PC) structures hold promise to address the aforementioned challenges in fluorescence enhancement. In this review, we provide a broad overview of PCs by explaining their structures, design strategies, fabrication techniques, and sensing principles. Furthermore, we discuss recent applications of PC-enhanced fluorescence-based biosensors incorporated with emerging technologies, including nucleic acids sensing, protein detection, and steroid monitoring. Finally, we discuss current challenges associated with PC-enhanced fluorescence and provide an outlook for fluorescence enhancement with photonic-plasmonics coupling and their promise for point-of-care biosensing as well monitoring analytes of biological and environmental relevance. The review presents the transdisciplinary applications of PCs in the broad arena of fluorescence spectroscopy with broad applications in photo-plasmonics, life science research, materials chemistry, cancer diagnostics, and internet of things. [ABSTRACT FROM AUTHOR]

The ability to self-test for HIV is vital to preventing transmission, particularly when used in concert with HIV biomedical prevention modalities, such as pre-exposure prophylaxis (PrEP). In this paper, we review recent developments in HIV self-testing and self-sampling methods, and the potential future impact of novel materials and methods that emerged through efforts to develop more effective point-of-care (POC) SARS-CoV-2 diagnostics. We address the gaps in existing HIV self-testing technologies, where improvements in test sensitivity, sample-to-answer time, simplicity, and cost are needed to enhance diagnostic accuracy and widespread accessibility. We discuss potential paths toward the next generation of HIV self-testing through sample collection materials, biosensing assay techniques, and miniaturized instrumentation. We discuss the implications for other applications, such as self-monitoring of HIV viral load and other infectious diseases. [ABSTRACT FROM AUTHOR]

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]

While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence. Nanoscale emitters are useful for measuring biomolecular interactions, but are limited by weak signals. Here, the authors use a photonic crystal surface for 3000-fold signal enhancement, achieving single emitter sensitivity with extended on-time, and demonstrate its application in miRNA biomarker sensing. [ABSTRACT FROM AUTHOR]

Interferometric scattering microscopy is increasingly employed in biomedical research owing to its extraordinary capability of detecting nano-objects individually through their intrinsic elastic scattering. To significantly improve the signal-to-noise ratio without increasing illumination intensity, we developed photonic resonator interferometric scattering microscopy (PRISM) in which a dielectric photonic crystal (PC) resonator is utilized as the sample substrate. The scattered light is amplified by the PC through resonant near-field enhancement, which then interferes with the <1% transmitted light to create a large intensity contrast. Importantly, the scattered photons assume the wavevectors delineated by PC's photonic band structure, resulting in the ability to utilize a non-immersion objective without significant loss at illumination density as low as 25 W cm−2. An analytical model of the scattering process is discussed, followed by demonstration of virus and protein detection. The results showcase the promise of nanophotonic surfaces in the development of resonance-enhanced interferometric microscopies. Here, the authors present photonic resonator interferometric scattering microscopy, which utilises a dielectric photonic crystal as the sample substrate. The resonant near-field enhancement leads to improved signal to noise ratio without increasing illumination intensity. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection. [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]

One-dimensional photonic crystal (PC) slabs are capable of enhancing the excitation of fluorescent material adsorbed on their surface. In this report, we demonstrate and verify by electromagnetic computer simulations that resonant leaky modes spectrally overlapping the laser wavelength used for fluorescent excitation are responsible for the enhanced excitation, and that the Q-factor of the PC resonance is proportional to the resonant electric field intensity and thus proportional to the fluorescent enhancement factor. As a demonstration, we have fabricated a single PC slab surface with an intentional spatial gradient in the resonant wavelength and demonstrate enhanced fluorescence only from locations on the PC surface with a leaky mode corresponding to a 633 nm HeNe laser used to excite Cyanine 5 dye deposited uniformly across the PC. The results show that enhanced fluorescence signals for one-dimensional PC slabs originate from increased excitation of the fluorescent dye. [ABSTRACT FROM AUTHOR]

We experimentally verify the near-field nature of the fluorescence enhancement afforded by large area planar photonic crystal resonators. This is done by mapping the fluorescence enhancement factor for a fluorophore as a function of its distance above the device surface and comparing the results to numerical calculations. Experiments show that the decay of the enhancement factor is exponential as a function of distance from the device surface, and excellent agreement with the numerically calculated exponential decay length is obtained. In contrast to approaches involving surface plasmons on metal surfaces, we find that the maximum enhancement is obtained when the fluorophore is closest to the photonic crystal surface due to the absence of quenching at short distances. We also show that the decay length is largely controlled by the wavelength of light resonant in the device. [ABSTRACT FROM AUTHOR]

Circulating exosomal microRNA (miR) represents a new class of blood-based biomarkers for cancer liquid biopsy. The detection of miR at a very low concentration and with single-base discrimination without the need for sophisticated equipment, large volumes, or elaborate sample processing is a challenge. To address this, we present an approach that is highly specific for a target miR sequence and has the ability to provide "digital" resolution of individual target molecules with high signal-to-noise ratio. Gold nanoparticle tags are prepared with thermodynamically optimized nucleic acid toehold probes that, when binding to a target miR sequence, displace a probe-protecting oligonucleotide and reveal a capture sequence that is used to selectively pull down the target-probe-nanoparticle complex to a photonic crystal (PC) biosensor surface. By matching the surface plasmon-resonant wavelength of the nanoparticle tag to the resonant wavelength of the PC nanostructure, the reflected light intensity from the PC is dramatically and locally quenched by the presence of each individual nanoparticle, enabling a form of biosensor microscopy that we call Photonic Resonator Absorption Microscopy (PRAM). Dynamic PRAM imaging of nanoparticle tag capture enables direct 100-aM limit of detection and single-base mismatch selectivity in a 2-h kinetic discrimination assay. The PRAM assay demonstrates that ultrasensitivity (<1 pM) and high selectivity can be achieved on a direct readout diagnostic. [ABSTRACT FROM AUTHOR]

We report on the adaptation of a smartphone’s rear-facing camera to function as a spectrometer that measures the spectrum of light scattered by common paper-based assay test strips. We utilize a cartridge that enables a linear series of test pads in a single strip to be swiped past the read head of the instrument while the phone’s camera records video. The strip is housed in a custom-fabricated cartridge that slides through the instrument to facilitate illumination with white light from the smartphone’s flash LED that is directed through an optical fiber. We demonstrate the ability to detect subtle changes in the scattered spectrum that enables quantitative analysis of single-analyte and multi-analyte strips. The demonstrated capability can be applied to broad classes of paper-based assays in which visual observation of colored strips is not sufficiently quantitative, and for which analysis of red-green-blue pixel values of a camera image are not capable of measuring complex scattered spectra. [ABSTRACT FROM AUTHOR]

We report on the implementation of an automated platform for detecting the presence of an antibody biomarker for human papillomavirus-associated oropharyngeal cancer from a single droplet of serum, in which a nanostructured photonic crystal surface is used to amplify the output of a fluorescence-linked immunosorbent assay. The platform is comprised of a microfluidic cartridge with integrated photonic crystal chips that interfaces with an assay instrument that automates the introduction of reagents, wash steps, and surface drying. Upon assay completion, the cartridge interfaces with a custom laser-scanning instrument that couples light into the photonic crystal at the optimal resonance condition for fluorescence enhancement. The instrument is used to measure the fluorescence intensity values of microarray spots corresponding to the biomarkers of interest, in addition to several experimental controls that verify correct functioning of the assay protocol. In this paper, we report both dose-response characterization of the system using anti-E7 antibody introduced at known concentrations into serum and characterization of a set of clinical samples from which results were compared with a conventional enzyme-linked immunosorbent assay performed in microplate format. The demonstrated capability represents a simple, rapid, automated, and high-sensitivity method for the multiplexed detection of protein biomarkers from a low-volume test sample. [ABSTRACT FROM PUBLISHER]

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]

The total analytical error of a photonic crystal (PC) biosensor in the determination of ferritin and soluble transferrin receptor (sTfR) as biomarkers of iron deficiency anemia in chronic kidney disease (CKD) patients was evaluated against certified ELISAs. Antigens were extracted from sera of CKD patients using functionalized iron-oxide nanoparticles (fAb-IONs) followed by magnetic separation. Immuno-complexes were recognized by complementary detection Ab affixed to the PC biosensor surface, and their signals were followed using the BIND instrument. Quantification was conducted against actual protein standards. Total calculated error (TEcalc) was estimated based on systematic (SE) and random error (RE) and compared against total allowed error (TEa) based on established quality specifications. Both detection platforms showed adequate linearity, specificity, and sensitivity for biomarkers. Means, SD, and CV were similar between biomarkers for both detection platforms. Compared to ELISA, inherent imprecision was higher on the PC biosensor for ferritin, but not for sTfR. High SE or RE in the PC biosensor when measuring either biomarker resulted in TEcalc higher than the TEa. This did not influence the diagnostic ability of the PC biosensor to discriminate CKD patients with low iron stores. The performance of the PC biosensor is similar to certified ELISAs; however, optimization is required to reduce TEcalc. [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 report the design, fabrication, and testing of a photonic crystal (PC) biosensor structure that incorporates a porous high refractive index TiO2 dielectric film that enables immobilization of capture proteins within an enhanced surface-area volume that spatially overlaps with the regions of resonant electromagnetic fields where biomolecular binding can produce the greatest shifts in photonic crystal resonant wavelength. Despite the nanoscale porosity of the sensor structure, the PC slab exhibits narrowband and high efficiency resonant reflection, enabling the structure to serve as a wavelength-tunable element of an external cavity laser. In the context of sensing small molecule interactions with much larger immobilized proteins, we demonstrate that the porous structure provides 3.7 x larger biosensor signals than an equivalent nonporous structure, while the external cavity laser (ECL) detection method provides capability for sensing picometer-scale shifts in the PC resonant wavelength caused by small molecule binding. The porous ECL achieves a record high figure of merit for label-free optical biosensors. [ABSTRACT FROM AUTHOR]

We demonstrate a method for combining sputtered TiO2 deposition with liquid phase dip-coating of a quantum dot (QD) layer that enables precise depth placement of QD emitters within a high-index dielectric film, using a photonic crystal (PC) slab resonator to demonstrate enhanced emission from the QDs when they are located at a specific depth within the film. The depth of the QDs within the PC is found to modulate the resonant wavelength of the PC as well as the emission enhancement efficiency, as the semiconducting material embedded within the dielectric changes its spatial overlap with the resonant mode. [ABSTRACT FROM AUTHOR]

In this study, a planar‐surface photonic crystal (PC) biosensor for quantitative, kinetic, label‐free imaging of cell–surface interactions is demonstrated. The planar biosensor surface eliminates external stimuli to the cells caused by substrate topography to more accurately reflect smooth surface environment encountered by many cell types in vitro. Here, a fabrication approach that combines nanoreplica molding and a horizontal dipping process is used to planarize the surface of the PC biosensor. The planar PC biosensor maintains a high detection sensitivity that enables the monitoring of live cell–substrate interactions with spatial resolution sufficient for observing intracellular attachment strength gradients and the extensions of filopodia from the cell body. The evolution of cell morphology during the attachment and spreading process of 3T3 fibroblast cells is compared between planar and grating‐structured PC biosensors. The planar surface effectively eliminates the directionally biased cellular attachment behaviors that are observed on the grating‐structured surface. This work represents an important step forward in the development of label‐free techniques for observing cellular processes without unintended external environmental modulation. [ABSTRACT FROM AUTHOR]

We review the development and application of nanostructured photonic crystal surfaces and a hyperspectral reflectance imaging detection instrument which, when used together, represent a new form of optical microscopy that enables label-free, quantitative, and kinetic monitoring of biomaterial interaction with substrate surfaces. Photonic Crystal Enhanced Microscopy (PCEM) has been used to detect broad classes of materials which include dielectric nanoparticles, metal plasmonic nanoparticles, biomolecular layers, and live cells. Because PCEM does not require cytotoxic stains or photobleachable fluorescent dyes, it is especially useful for monitoring the long-term interactions of cells with extracellular matrix surfaces. PCEM is only sensitive to the attachment of cell components within ∼200 nm of the photonic crystal surface, which may correspond to the region of most interest for adhesion processes that involve stem cell differentiation, chemotaxis, and metastasis. PCEM has also demonstrated sufficient sensitivity for sensing nanoparticle contrast agents that are roughly the same size as protein molecules, which may enable applications in "digital" diagnostics with single molecule sensing resolution. We will review PCEM's development history, operating principles, nanostructure design, and imaging modalities that enable tracking of optical scatterers, emitters, absorbers, and centers of dielectric permittivity. [ABSTRACT FROM AUTHOR]

Photonic crystal surfaces that are designed to function as wavelength-selective optical resonators have become a widely adopted platform for label-free biosensing, and for enhancement of the output of photon emitting tags used throughout life science research and in vitro diagnostics. While some applications, such as analysis of drug-protein interactions, require extremely high resolution and the ability to accurately correct for measurement artifacts, others require sensitivity that is high enough for detection of disease biomarkers in serum with concentrations $\sim 1$ pg/ml. As the analysis of cells becomes increasingly important for studying the behavior of stem cells, cancer cells, and biofilms under a variety of conditions, approaches that enable high-resolution imaging of live cells without cytotoxic stains or photobleachable fluorescent dyes are providing new tools to biologists who seek to observe individual cells over extended time periods. This paper will review several recent advances in photonic crystal biosensor detection instrumentation and device structures that are being applied toward direct detection of small molecules in the context of high-throughput drug screening, photonic crystal fluorescence enhancement as utilized for high sensitivity multiplexed cancer biomarker detection, and label-free high-resolution imaging of cells and individual nanoparticles as a new tool for life science research and single-molecule diagnostics. [ABSTRACT FROM PUBLISHER]

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/ NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts. [ABSTRACT FROM AUTHOR]

Tailored optical output, such as color purity and efficient optical intensity, are critical considerations for displays, particularly in mobile applications. To this end, we demonstrate a replica molded photonic crystal structure with embedded quantum dots. Electrohydrodynamic jet printing is used to control the position of the quantum dots within the device structure. This results in significantly less waste of the quantum dot material than application through drop-casting or spin coating. In addition, the targeted placement of the quantum dots minimizes any emission outside of the resonant enhancement field, which enables an 8 × output enhancement and highly polarized emission from the photonic crystal structure. [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 report a fundamentally new approach to enhance fluorescence in which surface adsorbed fluorophore-tagged biomolecules are excited on a photonic crystal surface that functions as a narrow bandwidth and tunable mirror of an external cavity laser. This scheme leads to ∼10× increase in the electromagnetic enhancement factor compared to ordinary photonic crystal enhanced fluorescence. In our experiments, the cavity automatically tunes its lasing wavelength to the resonance wavelength of the photonic crystal, ensuring optimal on-resonance coupling even in the presence of variable device parameters and variations in the density of surface-adsorbed capture molecules. We achieve ∼105× improvement in the limit of detection of a fluorophore-tagged protein compared to its detection on an unpatterned glass substrate. The enhanced fluorescence signal and easy optical alignment make cavity-coupled photonic crystals a viable approach for further reducing detection limits of optically-excited light emitters that are used in biological assays. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

We demonstrate the enhancement of a liquid-based homogenous fluorescence assay using the resonant electric fields from a photonic crystal (PC) surface. Because evanescent fields are confined to the liquid volume nearest to the photonic crystal, we developed a simple approach for integrating a PC fabricated on a silicon substrate within a fluid channel with submicron height, using electrohydrodynamic jet (e-jet) printing of a light-curable epoxy adhesive to define the fluid channel pattern. The PC is excited by a custom-designed compact instrument that illuminates the PC with collimated light that precisely matches the resonant coupling condition when the PC is covered with aqueous media. Using a molecular beacon nucleic acid fluorescence resonant energy transfer (FRET) probe for a specific miRNA sequence, we demonstrate an 8× enhancement of the fluorescence emission signal, compared to performing the same assay without exciting resonance in the PC detecting a miRNA sequence at a concentration of 62 nM from a liquid volume of only ∼20 nL. The approach may be utilized for any liquid-based fluorescence assay for applications in point-of-care diagnostics, environmental monitoring, or pathogen detection. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]