We report a single-step, room-temperature, five to ten minute SARS-CoV-2 saliva self-monitoring method that overcomes the limitations of existing approaches through the use of fluorophore-releasing Designer DNA Nanostructures (DDNs) that bind with the multivalent pattern of spike proteins on the exterior intact virions and an inexpensive smartphone-linked, pocket-size fluorimeter, called a “V-Pod” for its resemblance to an Apple AirPod™ headphone case. We characterize the V-Pod fluorimeter performance and the DDN-based assay to demonstrate a clinically relevant detection limit of 104 virus particles/mL for pseudo-typed WT SARS-CoV-2 and 105 virus particles/mL for real pathogenic variants, including Delta, Omicron, and D614g.
We report an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a two-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 RNA from the 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 instrument heats the compartments from below, while LEDs illuminate from above. Fluorescence generation in the compartments is dynamically monitored by a smartphone camera. We characterize the assay time and detection limits for detecting Zika RNA and gamma-irradiated 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. We show that dynamic image analysis reduces the time required to designate an assay as a positive test to 22 minutes, compared to ~30-45 minutes for conventional analysis of the average fluorescent intensity of the entire compartment. We achieve a total sample-to-answer time in the range of 17-32 minutes, while demonstrating a viral RNA detection as low as 2.70×102 copies/ul, and a gamma-irradiated virus of 103 virus particles in a single 12.5 microliter droplet blood sample.
By combining biosensor microscopy that provides high contrast for detecting individual biomolecules with novel biochemistry methods that can effectively turn each target molecule into many digitally-counted sensing events, it is possible to achieve attomolar-scale limits of detection for cancer-specific nucleic acid target molecules (miRNA and ctDNA) while simultaneously obtaining thousands-to-one selectivity against single base variants. The presentation will describe the use of photonic metamaterials and associated detection instruments to amplify optical absorption and fluorescence emission. The biodetection technology platforms are used to perform assays using nucleic acid strand displacement reactions and CRISPR/Cas chemistry to sense target biomarkers from complex media. The “amplify-then-digitize” approach represents a new and powerful paradigm for molecular diagnostics, compared to the “digitize-then amplify” approach utilized in methods such as droplet digital PCR (ddPCR). Overall, we seek simple, rapid, room temperature, single-step assay methods that can be operate with small, inexpensive, and robust detection systems for applications in point of care diagnostics, laboratory-based diagnostics, and life science research applications.
Rapid, simple, inexpensive, and sensitive self-testing for SARS-CoV-2 is expected to be an important element of controlling the ongoing COVID pandemic. We report a novel approach in which saliva is mixed at room temperature with a Designer DNA Nanostructure (DDN) engineered to create a net-like structure that positions an array of highly specific nucleic acid aptamer-quencher locks at the locations of the trimeric spike proteins. When the spike proteins selectively unlock aptamers on the DDN, fluorescent reporter molecules are unquenched, generating an intense and easily measured optical signal. The fluorescence intensity, proportional to the virus concentration, is detected by a battery-powered palm-sized fluorimeter, whose functions are managed wirelessly with a Bluetooth-linked smartphone. Because the single-step, room temperature, test is performed in a conventional 0.2 mL PCR tube that is inserted into the fluorimeter, which resembles an Apple AirPods™ headphone case, we call the technology (DDN+fluorimeter+App) a “V-Pod.” We show that DDNs are highly specific only for detection of SARS-CoV-2 in both its initial form as well as common variants. The approach achieves a detection limit of 10,000 genome copies/mL, consistent with laboratory-based PCR, while requiring only one reagent and a 5-10 minute incubation time with saliva. Because DDNs are inexpensively synthesized, structurally stable nucleic acid constructs, and the V-Pod instrument is comprised of inexpensive electronic and photonic components, the approach offers potential for rapid self-monitoring of viral infection with integrated capability for contact tracing and interaction with health services.
Interferometric scattering microscopy is a newly emerging alternative to fluorescence microscopy in biomedical research and diagnostic testing due to its ability to detect nano-objects such as individual proteins, extracellular vesicles, and virions individually through their intrinsic elastic light scattering. To improve the signal-to-noise ratio, we developed photonic resonator interferometric scattering microscopy (PRISM) in which a photonic crystal (PC) resonator is used 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 intensity contrast. Importantly, the scattered photons assume the wavevectors defined 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/cm2. We demonstrate virus and protein detection, including highly selective capture and counting of intact pseudotype SARS-CoV-2 from saliva with sensitivity equivalent to conventional nucleic acid tests. The results showcase the promise of nanophotonic surfaces in the development of resonance-enhanced interferometric microscopies, and as a single step, room temperature, and rapid viral detection technology.
Newly demonstrated advanced biosensor imaging technologies utilize the unique electromagnetic capabilities of photonic metamaterials to enhance the interaction between light and biological matter. The resulting capabilities address gaps in existing technologies for biomolecular analysis that rely upon enzymatic and chemical amplification, costly instrumentation, and complex assay protocols. Through amplification of the excitation/extraction efficiency of light emitting tags, absorption efficiency of nanoparticle tags, and scattering efficiency of biological analytes, technology platforms have been demonstrated that are capable of ultrasensitive, digital-resolution, room temperature, isothermal, rapid, and highly quantitative biomolecular analysis.
Approaches are demonstrated that enable mobile devices, such as smartphones, to function as spectrophotometers with equivalent performance to laboratory instruments for measuring any diagnostic test that generates a colored liquid, fluorescent liquid, or colored solid surface. We envision mobile health diagnostic applications in which smartphone integrated measurement of point-of-care assays enables smart service systems for efficiently connecting patients with health care providers and other health services. A key to this capability is to offer valid tests that are equivalent to those performed in the laboratory by utilizing the same reagents, experimental controls, and calibration standards as conventional assays.
KEYWORDS: Nanoantennas, Photonic crystals, Plasmonics, Nanoparticles, Resonance enhancement, Scanning electron microscopy, Biosensing, Absorption, Near field
Coupling of electromagnetic energy from macroscale external light sources to nanometer-scale volumes associated with plasmonic and dielectric nanoantennas is utilized for excitation of Raman scattering on nanoparticles, excitation of semiconductor quantum dots, and resonant optical absorption. These phenomena are used in the context of detection of biomolecules for applications that include disease diagnostics from single-droplet test samples derived from bodily fluids.
200 mm diameter wafer-scale fabrication, metrology, and optical modeling results are reviewed for surface plasmon resonance (SPR) sensors based on 2-D metallic nano-dome and nano-hole arrays (NHA's) as well as 1-D photonic crystal sensors based on a leaky-waveguide mode resonance effect, with potential applications in label free sensing, surface enhanced Raman spectroscopy (SERS), and surface-enhanced fluorescence spectroscopy (SEFS). Potential markets include micro-arrays for medical diagnostics, forensic testing, environmental monitoring, and food safety. 1-D and 2-D nanostructures were fabricated on glass, fused silica, and silicon wafers using optical lithography and semiconductor processing techniques. Wafer-scale optical metrology results are compared to FDTD modeling and presented along with application-based performance results, including label-free plasmonic and photonic crystal sensing of both surface binding kinetics and bulk refractive index changes. In addition, SEFS and SERS results are presented for 1-D photonic crystal and 2-D metallic nano-array structures. Normal incidence transmittance results for a 550 nm pitch NHA showed good bulk refractive index sensitivity, however an intensity-based design with 665 nm pitch was chosen for use as a compact, label-free sensor at both 650 and 632.8 nm wavelengths. The optimized NHA sensor gives an SPR shift of about 480 nm per refractive index unit when detecting a series of 0-40% glucose solutions, but according to modeling shows about 10 times greater surface sensitivity when operating at 532 nm. Narrow-band photonic crystal resonance sensors showed quality factors over 200, with reasonable wafer-uniformity in terms of both resonance position and peak height.
Here we demonstrate three principle modalities for a smartphone-based spectrometer: absorption, fluorescence, and photonic crystal (PC)-based label-free detection. When combined with some simple optical components, the rear-facing CMOS camera in a mobile device can provide spectrometric data that rivals that of laboratory instruments, but at a fraction of the cost. The use of a smartphone-based platform poses significant advantages based upon the rise of smartphone apps, which allow for user-interface and data-processing algorithms to be packaged and distributed within environments that are externally maintained with potential for integration with services such as cloud storage, GIS-tagging, and remote expert analysis. We demonstrate the absorption modality of our device by performing an enzyme-linked immunosorbent assay (ELISA) on both a cancer biomarker and a peanut allergen, demonstrating clinically relevant limits of detection (LOD). Second, we demonstrate the success of a molecular beacon (MB)-based assay on the smartphone platform, achieving an LOD of 1.3 pM for a specific RNA sequence, less than that of a commercial benchtop instrument. Finally, we use a PC biosensor to perform label-free detection of a representative biological interaction: Protein A and human immunoglobulin G (IgG) in the nanomolar regime. Our work represents the first demonstration of smartphone-based spectroscopy for biological assays, and the first mobile-device-enabled detection instrument that serves to measure three distinct sensing modalities (label-free biosensing, absorption spectroscopy, and fluorescence spectroscopy). The smartphone platform has the potential to expand the use of spectrometric analysis to environments assay from the laboratory, which may include rural or remote locations, low-resource settings, and consumer markets.
We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays.
We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a
specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also
demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable
biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.
Photonic crystal surfaces that can be fabricated inexpensively over large surface areas can be designed to produce optical
resonances for any desired wavelength in the optical spectrum from ultraviolet to infrared. Label-free biosensing is
obtained by measuring shifts in the resonant wavelength as biomaterial deposits on the photonic crystal, while the intensified electric fields that occur due to coupling of illumination at the resonant wavelength may be used to more
effectively excite fluorescence or Raman scattering. Photon emitters, such as quantum dots, fluorescent dye molecules,
and Raman scatters can efficiently couple their energy to detection instruments when they are in close proximity to a
photonic crystal with a resonance that matches the emission wavelength. Finally, the narrowband optical filtering capability of photonic crystals can be effectively applied for infrared absorption imaging of biological specimens at discrete wavelengths. This talk will summarize recent activities in the Nano Sensors Group at the University of Illinois
in which photonic crystals are used to address a variety of problems in biological sensing.
We demonstrated gold-coated polymer surface enhanced Raman scattering (SERS) substrates with a pair of complementary structures-positive and inverted pyramid array structures fabricated by a multiple-step molding and replication process. The uniform SERS enhancement factors over the entire device surface were measured as 7.2×104 for positive pyramid substrates while 1.6×106 for inverted pyramid substrates with Rhodamine 6G as the target analyte. Based on the optical reflection measurement and finite difference time domain simulation result, the enhancement factor difference is attributable to plasmon resonance matching and to SERS "hot spots" distribution. With this simple, fast, and versatile complementary molding process, we can produce polymer SERS substrates with extremely low cost, high throughput, and high repeatability.
Optical resonances produced by photonic crystal surfaces can be designed to substantially amplify the
electric field excitation of fluorophores while at the same time increasing the collection efficiency of
emitted photons. Because PC surfaces can be produced inexpensively over large areas by replica molding,
they offer an effective means for increasing sensitivity for broad classes of surface-based fluorescent
assays. In this talk, the design and fabrication of photonic crystal surfaces and detection instrumentation
for fluorescent enhancement will be described, along with demonstrated applications in gene expression
microarrays and protein biomarker microarrays.
Photonic crystals are fabricated on plastic surfaces, producing narrow bandwidth resonances at any desired wavelength.
While shifts in the resonant wavelength quantify the density of adsorbed biomaterial, the resonances also enhance the
output of adsorbed fluorophores. The combined attributes of photonic crystals enable highly sensitive label-free
detection and greatly amplified sensitivity of any fluorescence-based assay for applications in life science research, drug
discovery, and environmental pathogen detection. Applications for label-free selective detection of viral pathogens with
sensitivity of ~30 focal forming units, and detection of spore pathogens with single-unit resolution are highlighted.
Amplified fluorescence, meanwhile, enables gene expression detection of DNA at concentrations ~2 orders of magnitude
lower than detection on optically passive surfaces.
Assays used in pharmaceutical research require a system that can not only detect biochemical interactions with high
sensitivity, but that can also perform many measurements in parallel while consuming low volumes of reagents. While
nearly all label-free biosensor transducers to date have been interfaced with a flow channel, the liquid handling system is
typically aligned and bonded to the transducer for supplying analytes to only a few sensors in parallel.
In this presentation, we describe a fabrication approach for photonic crystal biosensors that utilizes nanoreplica molding
to produce a network of sensors that are automatically self-aligned with a microfluidic network in a single process step.
The sensor/fluid network is inexpensively produced on large surface areas upon flexible plastic substrates, allowing the
device to be incorporated into standard format 96-well microplates. A simple flow scheme using hydrostatic pressure
applied through a single control point enables immobilization of capture ligands upon a large number of sensors with
220 nL of reagent, and subsequent exposure of the sensors to test samples. A high resolution imaging detection
instrument is capable of monitoring the binding within parallel channels at rates compatible with determining kinetic
binding constants between the immobilized ligands and the analytes. The first implementation of this system is capable
of monitoring the kinetic interactions of 11 flow channels at once, and a total of 88 channels within an integrated
biosensor microplate in rapid succession. The system was initially tested to characterize the interaction between sets of
proteins with known binding behavior.
This presentation will describe the design, fabrication, and testing of 2-element, and 8-element micro-Chemical Analysis Array ((mu) CANARY) sensor chips and address electronics. The detailed performance characterization of (mu) CANARY sensors for vapor and liquid phase detection will be presented. For vapor detection, NRL has applied polymer receptor coatings targeted at detection of chemical weapon agents, and has performed extensive chemical vapor exposure tests using two chemical weapon simulants and four vapor phase interferents. Data describing temperature dependence, long term/short term drift stability, detection limits, detection linearity, and vapor selectivity will be presented.
Assessing the medical condition of battlefield personnel requires the development of rapid, portable biological diagnostic assays for a wide variety of antigens and enzymes. Ideally, such an assay would be inexpensive, small, and require no added reagents while maintaining the sensitivity and accuracy of laboratory-based assays. In this work, a microelectromechanical (MEMS) based biological assay sensor is presented which is expected to meet the above requirements. The sensor is a thin silicon membrane resonator (SMR) which registers a decrease in resonant frequency when mass is adsorbed onto its surface. By coating the sensor surface with a monolayer of antibody, for example, we have detected the corresponding antigen with a detection resolution of 0.25 ng/ml in phosphate buffer solution. Micromachining techniques are being used to integrate many (64 elements on the first test chip) identical SMR sensors into a single silicon chip which would be capable of simultaneously performing a wide variety of biomedical assays. The sensors require only a small printed circuit board and 8V power supply to operate and provide a readout. The presentation will describe the operation of the SMR sensor, the fabrication of the sensor array, and initial test results using commercially available animal immunoglobulins in laboratory-prepared test solutions.
Small, low cost microphones with high sensitivity at frequencies greater than 20 KHz are desired for applications such as ultrasonic imaging and communication links. To minimize stray capacitance between the microphone and its amplifier circuit, process compatibility between the microphone and on-chip circuitry is also desired to facilitate integration. In this work, we have demonstrated micromachined microphones packaged with hybrid JFET amplifier circuitry with frequency response extending to 100 KHz, and voltage sensitivity of approximately 2.0 mV/Pa from 100 Hz to 10 KHz, and 16.5 mV/Pa at 30 KHz with a bias voltage of 8.0 V. The microphones are fabricated with membranes and fixed backplates made of low temperature plasma-enhanced chemical vapor deposited (PECVD) silicon nitride. Because the maximum temperature of the fabrication process is 300 degrees Celsius, microphones may be built on silicon wafers from any commercial CMOS foundry without affecting transistor characteristics, allowing integration with sophisticated amplifier circuitry. Low stress silicon nitride deposition was used to produce membranes up to 2.0 mm diameter and 0.5 micrometer thickness with plus or minus 0.10 micrometer flatness. The excellent planarity of both the diaphragm and the backplate, combined with a narrow sense gap (approximately 2 micrometers) results in high output capacitance (up to 6.0 pF). The high output capacitance results in noise spectral density which is approximately 3x lower than silicon diaphragms microphones previously fabricated by the authors. Diaphragms with corrugations were fabricated to relive tensile stress, to increase deflection per unit pressure and to increase deflection linearity with pressure.
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