Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2.

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 details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/µL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/µL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.

Nanopatterning reconfigurable magnetic landscapes via thermally assisted scanning probe lithography.

The search for novel tools to control magnetism at the nanoscale is crucial for the development of new paradigms in optics, electronics and spintronics. So far, the fabrication of magnetic nanostructures has been achieved mainly through irreversible structural or chemical modifications. Here, we propose a new concept for creating reconfigurable magnetic nanopatterns by crafting, at the nanoscale, the magnetic anisotropy landscape of a ferromagnetic layer exchange-coupled to an antiferromagnetic layer. By performing localized field cooling with the hot tip of a scanning probe microscope, magnetic structures, with arbitrarily oriented magnetization and tunable unidirectional anisotropy, are reversibly patterned without modifying the film chemistry and topography. This opens unforeseen possibilities for the development of novel metamaterials with finely tuned magnetic properties, such as reconfigurable magneto-plasmonic and magnonic crystals. In this context, we experimentally demonstrate spatially controlled spin wave excitation and propagation in magnetic structures patterned with the proposed method.

Note: electrical and thermal characterization of a ferroelectric thin film with an electro-thermal nanoprobe.

The localized temperature-dependent piezoelectric response of ferroelectric barium strontium titanate (BST) thin films is studied using an electro-thermal (ET) nanoprobe. The ET probe provides independent electrical and thermal excitation to a nanometer-scale volume of the specimen and is capable of detecting the phase transition temperature of the BST thin films. The piezoresponse measured by the ET probe follows the temperature dependence of the piezoelectric constant, whereas with bulk heating the response follows the temperature dependence of the spontaneous polarization. The observed differences stem from the localized inhomogeneous electro-thermal field distribution at the specimen.

Infrared microspectroscopy combined with conventional atomic force microscopy.

This paper reports nanotopography and mid infrared (IR) microspectroscopic imaging coupled within the same atomic force microscope (AFM). The reported advances are enabled by using a bimaterial microcantilever, conventionally used for standard AFM imaging, as a detector of monochromatic IR light. IR light intensity is recorded as thermomechanical bending of the cantilever measured upon illumination with intensity-modulated, narrowband radiation. The cantilever bending is then correlated with the sample's IR absorption. Spatial resolution was characterized by imaging a USAF 1951 optical resolution target made of SU-8 photoresist. The spatial resolution of the AFM topography measurement was a few nanometers as expected, while the spatial resolution of the IR measurement was 24.4 µm using relatively coarse spectral resolution (25-125 cm(-1)). In addition to well-controlled samples demonstrating the spatial and spectral properties of the setup, we used the method to map engineered skin and three-dimensional cell culture samples. This research combines modest IR imaging capabilities with the exceptional topographical imaging of conventional AFM to provide advantages of both in a facile manner.

High-sensitivity nanometer-scale infrared spectroscopy using a contact mode microcantilever with an internal resonator paddle.

Infrared (IR) spectroscopy is one of the most widely used techniques for identifying and characterizing materials, but is diffraction limited to a spatial resolution of no smaller than several micrometers. This paper reports IR spectroscopy with 100 nm spatial resolution, using a tunable laser whose absorption in an organic layer is measured via atomic force microscopy. Wavelength-dependent absorption in the sample results in local thermomechanical deformation, which is sensed using the sharp tip of a resonant atomic force microscope cantilever. We introduce a cantilever and system design capable of 100 nm spatial resolution and a 6 x sensitivity improvement over previous approaches.

A 100 nanometer scale resistive heater-thermometer on a silicon cantilever.

This paper reports a method for fabricating a 100 nm scale heater-thermometer into a silicon microcantilever based on contact photolithography and a controlled annealing process. The heater is formed during a photolithography process that can achieve a minimum feature size of about 1 microm, while careful control of doping and annealing parameters allows the heater size to be further decreased, to a width of 100 nm. The heater is fabricated onto the free end of a silicon cantilever suitable for scanning probe microscopy, and can be integrated into cantilevers with or without sharp tips. The fabricated heater has a maximum temperature of over 700 degrees C, and a heating time of 56 micros to reach 500 degrees C.

Measuring material softening with nanoscale spatial resolution using heated silicon probes.

This article describes the use of heated silicon atomic force microscopy probes to perform local thermal analysis (LTA) of a thin film of polystyrene. The experiments measure film softening behavior with 100 nm spatial resolution, whereas previous research on LTA used probes that had a resolution near 10 microm, which was too large to investigate some types of features. This article demonstrates four methods by which heated silicon probes can perform thermal analysis with nanoscale spatial resolution. The polystyrene softening temperature measured from nanoscale LTA techniques is 120 degrees C, compared to 100 degrees C, measured with bulk ellipsometry. The discrepancy is attributed to the thermal contact resistance at the end of the silicon probe tip, on the order of 10(7)K/W, which modulates heat flow between the tip and sample and governs the fundamental limits of this technique. The use of a silicon probe for LTA enables bulk fabrication, parallelization for high-throughput analysis, and fabrication of a sharp tip capable of nanoscale spatial resolution.

Advanced temperature compensation for piezoresistive sensors based on crystallographic orientation.

We describe a highly effective method of reducing thermal sensitivity in piezoresistive sensors, in particular silicon cantilevers, by taking advantage of the dependence of the piezoresistive coefficient of silicon on crystallographic orientation. Two similar strain-sensing elements are used, positioned at 45 degrees to each other: One is set along a crystalline axis associated with a maximum piezoresistive coefficient to produce the displacement signal, while the other is set along an axis of the vanishing coefficient to produce the reference signal. Unlike other approaches, both sensing elements are coupled to the same cantilever body, maximizing thermal equilibration. Measurements show at least one order of magnitude improvement in thermal disturbance rejection over conventional approaches using uncoupled resistors.

The use of low-dose histamine therapy in otolaryngology.

Low-dose histamine therapy has been prescribed by otolaryngologists primarily to treat Bell's palsy, vestibular disorders, vascular headache, Ménière's attacks, and urticaria vasculitis. The scientific explanations for the usefulness of this once-empiric treatment are becoming more apparent. Two methods of establishing the appropriate dosages have emerged: the empiric, optimum-dosage approach and the objective, endpoint-titration approach. In this article, the author describes and recommends the latter approach. The author also reports on a 100-patient retrospective clinical study that revealed that the objective, endpoint-titration approach was effective in treating 80% of patients. In light of such success and the ease and economy of this treatment, low-dose histamine therapy appears to be a valuable clinical tool.

Alternatives in the diagnosis and treatment of food allergies.

The diagnosis of food allergy remains a difficult and often inexact proposition, with the treatment being equally cumbersome and complex. For this reason a variety of approaches have been developed, varying in acceptance and approval. Although many approaches represent alternative medicine, today's alternative may be tomorrow's standard.

Food hypersensitivity in otolaryngology. Manifestations, diagnosis, and treatment.

Diet hypersensitivity may be "allergic," that is, caused by some form of immunologic reaction, or nonallergic. The latter includes chemical pharmacologic, enzymatic, or dietary disorders. Food allergies may be immediate Gell and Coombs type I reactions (5%), or delayed "serum sickness" type reactions (95%). The latter type is influenced by frequency of exposure. Any inflammation of the intestinal tract allows abnormal absorption of more allergenic food megamolecules. Manifestations of food allergy seen frequently by the otolaryngologist are legion, afflicting all areas of the discipline's concern. Diagnostic techniques rely on singly or some combination of history taking, challenge testing, and skin testing. The intracutaneous progressive dilution food test is the standardized skin test procedure now taught by the American Academy of Otolaryngic Allergy. Treatment techniques include specific dietary abstinence, a rotary diversified diet, and neutralization immunotherapy.

The intracutaneous progressive dilution multi-food test.

Data have been published supporting the view that skin response is more valid and reliable than symptom provocation for interpretation of the intracutaneous provocative food test (IPFT). As such, an intracutaneous progressive dilution food test technique interpreted by skin response is now the standard IPFT technique taught at American Academy of Otolaryngic Allergy-sponsored courses. While this test technique is less time-consuming than the 5-day oral challenge food test of Rinkel, the 15- to 90-minute per test demand still presents a significant deterrant to widespread use. Here we present a time-efficient, multi-test technique that allows testing of three to seven foods at one time. The technique is not recommended for use on patients with a history of severe allergic reactions, such as asthma or angioedema, or for testing any food that by history may produce an anaphylactic reaction.

The evolution of otolaryngic allergy practices.

We review the evolution of the allergy diagnosis and treatment techniques endorsed by the American Academy of Otolaryngic Allergy. These endorsements were made only after careful consideration of supporting data accumulated during their evolution.

Efficacy of alternative tests for delayed-cyclic food hypersensitivity.

With the oral challenge food test (OCFT) used as the standard for delayed-cyclic food hypersensitivity diagnosis, blinded comparison studies were accomplished with 175 in vitro food specific IgE and 180 IgG radioallergosorbent tests, 180 food enzyme-linked immune complex assays, and 155 in vivo Multi-Test prick tests. The study was multi-centered, eight physicians and 37 patients participating. All of the compared tests were shown to be approximately 50% efficient when compared with the OCFT results, and, thus, nonefficacious.

Clinical significance of molds newly available for radioallergosorbent testing.

Three years of weekly Rotorod airborne allergen reports from Corpus Christi, Texas, were reviewed. Five of 10 newly available airborne molds for radioallergosorbent testing (RAST) were present in significant numbers. Three hundred seventy-five mold RAST evaluations were expanded to include the five old and these five new molds. Except for Alternaria, the frequency of and allegenic response to the new molds were generally greater than those for the five old molds. The usual Alternaria-Cladosporium mold screen proved less satisfactory when these five new molds were added to the mold battery; however, the addition of Helminthosporium to the screen corrected this deficiency. Cross-reactivity production of false-positive responses seems unlikely because of frequent significant variance in RAST scores from mold to mold in the same patient.

Provocation-neutralization: a two-part study. Part I. The intracutaneous provocative food test: a multi-center comparison study.

This study investigated the clinical usefulness of the intracutaneous provocative-neutralization food test (IPFT). Thirty-seven patients were tested for five identical food allergies by eight physicians in different geographical locations. Throughout the study, comparison was made between the IPFT when interpreted by skin response (IPFT SK) and when interpreted by symptom provocation (IPFT PR). Double-blind IPFT results were compared with those of previously accomplished oral challenge food tests (OCFT). IPFT reliability was determined by a double-blind comparison of the initial IPFT, with two subsequent IPFTs performed 7 days apart. Correlation of the IPFT SK and IPFT PR with the OCFT provided validity coefficients of 0.78 and 0.61 respectively, both significant beyond the 0.01 level of confidence. Reliability of the IPFT SK and IPFT PR was shown to be 0.68 and 0.40, respectively. The IPFT SK was significant beyond the 0.01 level of confidence and the IPFT PR was significant beyond the 0.05 level of confidence.

Provocation-neutralization: a two-part study. Part II. Subcutaneous neutralization therapy: a multi-center study.

Presented is a triple-blind crossover study that investigates the efficacy of subcutaneous neutralization food hypersensitivity therapy. Seven physicians and thirty-three patients from various parts of the country participated. Each patient underwent three 2-week treatment sessions, with 1 week off treatment between each session. During each treatment session, one injection a day was given. The injection consisted of a placebo for one 2-week session, and the active allergen during the other two sessions. The active dose was determined by earlier intracutaneous provocative food testing. The diet during the study period was not varied. Medication-symptom diaries were maintained and treatment result evaluations for both individual complaints and overall results were detailed on a standard form at the end of each treatment session. While the number of foods treated per patient varied from 1 to 13, the majority were treated with 3 to 5 foods. Treatment with the active medication was more efficacious than with placebo. A few patients' symptoms were aggravated with the active medication. This indicates a correct diagnosis, but incorrect treatment dose. In the clinical setting such adverse response should be reversed. Overall, neutralization subcutaneous treatment should be beneficial approximately 75% of the time, and further enhanced by supplemental diet manipulation.

Allergic disorders in the otolaryngologic practice.

The diagnosis and treatment of otolaryngic allergy is an integral part of the specialty of otolaryngology. By combining special medical and surgical expertise, the otolaryngologist should be able to more effectively manage the many allergic afflictions of the ear, nose, throat, head, and neck areas. At the very minimum, the otolaryngologist must appreciate the importance of otolaryngic allergy diagnosis and management and include allergy in the differential diagnoses where appropriate. If the otolaryngologist is professionally prepared and physically equipped to manage such problems, the patient will profit.

Efficacy of a screening radioallergosorbent test.

Controversy over the routine use of the radioallergosorbent test (RAST) for initial inhalant allergy diagnosis largely concerns expense to the patient. Presented is a method for utilizing the RAST, with all its inherent advantages, at a competitive cost to both negatively and positively responding patients. The initial use of a "screening" RAST that utilizes approximately one third the number of tests in the usual full RAST battery is recommended. When all responses on the screening RAST battery are negative, further RAST testing proves generally unnecessary. For positive responders, the screening RAST often eliminates the need of additional pollen and/or mold allergen testing, a further cost-containment feature. The presented study illustrates the efficacy of this technique. A background section is provided to familiarize the reader with the common modalities available for testing and treating IgE-mediated inhalant allergies.