Validation of a molecular assay to detect SARS-CoV-2 in saliva.

AIM: To validate a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay to detect SARS-CoV-2 in saliva in two independent Aotearoa New Zealand laboratories. METHODS: An RT-qPCR assay developed at University of Illinois Urbana-Champaign, USA, was validated in two New Zealand laboratories. Analytical measures, such as limit of detection (LOD) and cross-reactivity, were performed. One hundred and forty-seven saliva samples, each paired with a contemporaneously collected nasal swab, mainly of nasopharyngeal origin, were received. Positive (N=33) and negative (N=114) samples were tested blindly in each laboratory. Diagnostic sensitivity and specificity were then calculated. RESULTS: The LOD was <0.75 copy per µL and no cross-reactivity with MERS-CoV was detected. There was complete concordance between laboratories for all saliva samples with the quantification cycle values for all three genes in close agreement. Saliva had 98.7% concordance with paired nasal samples: and a sensitivity, specificity and accuracy of 97.0%, 99.1% and 99.1%, respectively. CONCLUSION: This saliva RT-qPCR assay produces reproducible results with a low LOD. High sensitivity and specificity make it a reliable option for SARS-CoV-2 testing, including for asymptomatic people requiring regular screening.

Spatial extent of cochlear infrared neural stimulation determined by tone-on-light masking.

Artificial neural stimulation is widely used in clinic, rehabilitation, and research. One of the limitations of electrical stimulation is the current spread in tissue. Recently, pulsed mid-infrared laser stimulation of nerves has been investigated as an alternative stimulation method. The likely benefits of infrared neural stimulation (INS) include spatial selectivity of stimulation, noncontact mode of operation, and the lack of stimulation artifact in simultaneous electrical recordings. The hypothesis for this study is that INS of the cochlear spiral ganglion at low pulse energy is as spatially selective as low-level tonal stimulation of the cochlea. Spatial selectivity was measured using a masking method. An optical pulse with fixed optical parameters was delivered through a 200-µm diameter optical fiber. An acoustic tone, variable in frequency and level, was presented simultaneously with the optical pulse. Tone-on-light masking in gerbils revealed tuning curves with best frequencies between 5.3 and 11.4 kHz. The width of the tone-on-light tuning curves was similar to the width of tone-on-tone tuning curves. The results indicate that the spatial area of INS in the gerbil cochlea is similar to the cochlear area excited by a low level acoustic tone, showing promising results for future use of INS in implantable cochlear prostheses.

In vivo, transcutaneous glucose sensing using surface-enhanced spatially offset Raman spectroscopy: multiple rats, improved hypoglycemic accuracy, low incident power, and continuous monitoring for greater than 17 days.

This paper presents the latest progress on quantitative, in vivo, transcutaneous glucose sensing using surface enhanced spatially offset Raman spectroscopy (SESORS). Silver film over nanosphere (AgFON) surfaces were functionalized with a mixed self-assembled monolayer (SAM) and implanted subcutaneously in Sprague-Dawley rats. The glucose concentration was monitored in the interstitial fluid of six separate rats. The results demonstrated excellent accuracy and consistency. Remarkably, the root-mean-square error of calibration (RMSEC) (3.6 mg/dL) and the root-mean-square error of prediction (RMSEP) (13.7 mg/dL) for low glucose concentration (<80 mg/dL) is lower than the current International Organization Standard (ISO/DIS 15197) requirements. Additionally, our sensor demonstrated functionality up 17 days after implantation, including 12 days under the laser safety level for human skin exposure with only one time calibration. Therefore, our SERS based sensor shows promise for the challenge of reliable continuous glucose sensing systems for optimal glycemic control.

Neural stimulation with optical radiation.

This paper reviews the existing research on infrared neural stimulation, a means of artificially stimulating neurons that has been proposed as an alternative to electrical stimulation. Infrared neural stimulation (INS) is defined as the direct induction of an evoked potential in response to a transient targeted deposition of optical energy. The foremost advantage of using optical radiation for neural stimulation is its spatial resolution. Exogenously applied or trans-genetically synthesized fluorophores are not used to achieve stimulation. Here, current work on INS is presented for motor nerves, sensory nerves, central nervous system, and in vitro preparations. A discussion follows addressing the mechanism of INS and its potential use in neuroprostheses. A brief review of neural depolarization involving other optical methods is also presented. Topics covered include optical stimulation concurrent with electrical stimulation, optical stimulation using exogenous fluorophores, and optical stimulation by transgenic induction of light-gated ion channels.

Transcutaneous glucose sensing by surface-enhanced spatially offset Raman spectroscopy in a rat model.

This letter presents the first quantitative, in vivo, transcutaneous glucose measurements using surface enhanced Raman spectroscopy (SERS). Silver film over nanosphere (AgFON) surfaces were functionalized with a mixed self-assembled monolayer (SAM) and implanted subcutaneously in a Sprague-Dawley rat. The glucose concentration was monitored in the interstitial fluid. SER spectra were collected from the sensor chip through the skin using spatially offset Raman spectroscopy (SORS). The combination of SERS and SORS is a powerful new approach to the challenging problem of in vivo metabolite and drug sensing.

Prediction range estimation from noisy Raman spectra with robust optimization.

Inferences need to be drawn in biological systems using experimental multivariate data. The number of samples collected in many such experiments is small, and the data are noisy. We present and study the performance of a robust optimization (RO) model for such situations. We adapt this model to generate a minimum and a maximum estimation of analyte concentration for a given sample, producing a prediction range. The calibration model was applied to sets of Raman spectra. In particular we used normal Raman measurements of pyridine/deuterated pyridine mixtures and spectra from a more complex glucose detection system based on surface-enhanced Raman spectroscopy. The results from the RO model were compared with prediction intervals estimated from partial least squares (PLS) method. We find that the RO prediction ranges included the actual concentration value of the sample more consistently than the 99% prediction intervals built with PLS methods.

In vivo characterization of human pigmented lesions by degree of linear polarization image maps using incident linearly polarized light.

BACKGROUND AND OBJECTIVE: Melanoma is the most serious form of skin cancer and often appears as an evolving multicolored skin growth. It is well documented that pre-existing atypical or dysplastic nevi can evolve into a melanoma. The development of an in vivo imaging system to characterize benign and malignant nevi has been emphasized to aid in early detection of melanoma. The goal of this study is to utilize a novel Stokes polarimetry imaging (SPI) system for the characterization of pigmented lesions, and to evaluate the SPI system in comparison to dermoscopy and histology images. STUDY DESIGN/MATERIALS AND METHODS: Linearly polarized light with varying incident polarization angles (IPA) illuminated various types of pigmented lesions. The melanocytic nesting patterns of pigmented lesions were characterized by constructing the degree-of-linear-polarization (DOLP) image map with comparison to dermoscopy and histology. The incident polarized light was filtered by visible filters for spectral imaging and incident deeply penetrating red light was used to correlate the SPI image with histopathological examination. RESULTS: The DOLP images with varying IPA at different visible wavelengths were used to characterize various kinds of pigmented lesions by showing subsurface melanocytic nesting distribution as well as morphological information with better resolution and contrast. In correlation with dermoscopy and histology, various defining features such as compound, junctional, lentiginous, reticular, globular patterns of melanocytic nests were identified. CONCLUSION: When imaging pigmented melanocytic lesions, the SPI system with varying IPA at the red light wavelength can better define the melanocytic nesting patterns in both the dermal epidermal junction and the dermis. The SPI system has the potential to be an effective in vivo method of detecting pre-malignant nevi and melanoma.

Progress toward an in vivo surface-enhanced Raman spectroscopy glucose sensor.

BACKGROUND: In this report, we detail our current work towards developing a surface-enhanced Raman spectroscopy (SERS) based sensor for in vivo glucose detection. Despite years of innovations in the development of blood glucose monitors, there remains a need for accurate continuous glucose sensors to provide care to rising numbers of diagnosed diabetes patients and mitigate secondary health complications associated with this metabolic disorder. METHODS: SERS is a highly specific and sensitive optical technique suitable for direct detection of glucose. The SERS effect is highly distance dependent, thus the glucose molecules need to be within a few nanometers or adsorbed to an SERS-active surface. In our sensor, this is achieved with a self-assembled monolayer (SAM) that facilitates reversible interactions between glucose molecules and the surface. The amount of glucose near the surface is proportional to its concentration in the surrounding environment. RESULTS: We determined that the SAM-functionalized surface is stable for at least 10 days and provides rapid, reversible partitioning. In vitro experiments in bovine plasma as well as in vivo experiments in rats demonstrated quantitative detection. CONCLUSIONS: We show successful use of the SERS glucose sensor in rats, making it the first in vivo SERS sensor. Furthermore, we demonstrate free space transdermal detection of a SERS signal through the rat's skin as an initial step toward developing a transcutaneous sensor.

Optical stimulation of auditory neurons: effects of acute and chronic deafening.

In developing neural prostheses, particular success has been realized with cochlear implants. These devices bypass damaged hair cells in the auditory system and electrically stimulate the auditory nerve directly. In contemporary cochlear implants, however, the injected electric current spreads widely along the scala tympani and across turns. Consequently, stimulation of spatially discrete spiral ganglion cell populations is difficult. In contrast to electrical stimulation, it has been shown that extremely spatially selective stimulation is possible using infrared radiation (e.g. [Izzo, A.D., Su, H.S., Pathria, J., Walsh Jr., J.T., Whitlon, D.S., Richter, C.-P., 2007a. Selectivity of neural stimulation in the auditory system: a comparison of optic and electric stimuli. J. Biomed. Opt. 12, 1-7]). Here, we explore the correlation between surviving spiral ganglion cells, following acute and chronic deafness induced by neomycin application into the middle ear, and neural stimulation using optical radiation and electrical current. In vivo experiments were conducted in gerbils. Before the animals were deafened, acoustic thresholds were obtained and neurons were stimulated with optical radiation at various pulse durations, radiation exposures, and pulse repetition rates. In one group of animals, measurements were made immediately after deafening, while the other group was tested at least four weeks after deafening. Deafness was confirmed by measuring acoustically evoked compound action potentials. Optically and electrically evoked compound action potentials and auditory brainstem responses were determined for different radiation exposures and for different electrical current amplitudes, respectively. After completion of the experiments, the animals were euthanized and the cochleae were harvested for histology. Acoustically evoked compound action potential thresholds were elevated by more than 40 dB after neomycin application in acutely deaf and more than 60 dB in chronically deaf animals. Compound action potential thresholds, which were determined with optical radiation pulses, were not significantly elevated in acutely deaf animals. However, in chronically deaf animals optically evoked CAP thresholds were elevated. Changes correlated with the number of surviving spiral ganglion cells and the optical parameters that were used for stimulation.

Laser stimulation of auditory neurons: effect of shorter pulse duration and penetration depth.

We have pioneered what we believe is a novel method of stimulating cochlear neurons, using pulsed infrared radiation, based on the hypothesis that optical radiation can provide more spatially selective stimulation of the cochlea than electric current. Very little of the available optical parameter space has been used for optical stimulation of neurons. Here, we use a pulsed diode laser (1.94 microm) to stimulate auditory neurons of the gerbil. Radiant exposures measured at CAP threshold are similar for pulse durations of 5, 10, 30, and 100 micros, but greater for 300-micros-long pulses. There is evidence that water absorption of optical radiation is a significant factor in optical stimulation. Heat-transfer-based analysis of the data indicates that potential structures involved in optical stimulation of cochlear neurons have a dimension on the order of approximately 10 microm. The implications of these data could direct further research and design of an optical cochlear implant.

Lactate and sequential lactate-glucose sensing using surface-enhanced Raman spectroscopy.

Lactate production under anaerobic conditions is indicative of human performance levels, fatigue, and hydration. Elevated lactate levels result from several medical conditions including congestive heart failure, hypoxia, and diabetic ketoacidosis. Real-time detection of lactate can therefore be useful for monitoring these medical conditions, posttrauma situations, and in evaluating the physical condition of a person engaged in strenuous activity. This paper represents a proof-of-concept demonstration of a lactate sensor based on surface-enhanced Raman spectroscopy (SERS). Furthermore, it points the direction toward a multianalyte sensing platform. A mixed decanethiol/mercaptohexanol partition layer is used herein to demonstrate SERS lactate sensing. The reversibility of the sensor surface is characterized by exposing it alternately to aqueous lactate solutions and buffer without lactate. The partitioning and departitioning time constants were both found to be approximately 30 s. In addition, physiological lactate levels (i.e., 6-240 mg/dL) were quantified in phosphate-buffered saline medium using multivariate analysis with a root-mean-square error of prediction of 39.6 mg/dL. Finally, reversibility was tested for sequential glucose and lactate exposures. Complete partitioning and departitioning of both analytes was demonstrated.

Optical stimulation of the facial nerve: a new monitoring technique?

OBJECTIVES/ HYPOTHESIS: One sequela of skull base surgery is iatrogenic damage to cranial nerves, which can be prevented if the nerve is identified. Devices that stimulate nerves with electric current assist in nerve identification. Contemporary devices have two main limitations: 1) the physical contact of the stimulating electrode and (2) the spread of the current through the tissue. In contrast to electrical stimulation, pulsed infrared optical radiation can be used to safely and selectively stimulate neural tissue and might be valuable for screening. METHODS: The gerbil facial nerve was exposed to 250 microsecond pulses of 2.12 microm radiation delivered via a 600-microm-diameter optical fiber at a repetition rate of 2 Hz. With use of 27 GA, 12-mm intradermal electrodes, muscle action potentials were recorded. Nerve samples were examined for possible tissue damage. RESULTS: Eight facial nerves were stimulated with radiant exposures between 0.71 and 1.77 J/cm, resulting in compound muscle action potentials (CmAPs) that were simultaneously measured at the m. orbicularis oculi, m. levator nasolabialis, and m. orbicularis oris. Resulting CmAP amplitudes were 0.3 to 0.4 mV, 0.15 to 1.4 mV, and 0.3 to 2.3 mV, respectively, depending on the radial location of the optical fiber and the radiant exposure. Individual nerve branches were also stimulated, resulting in CmAP amplitudes between 0.2 and 1.6 mV. Histology revealed tissue damage at radiant exposures of 2.2 J/cm but no apparent damage at radiant exposures of 2.0 J/cm. CONCLUSIONS: The experiments showed that selective muscle action potentials can be evoked optically in the gerbil facial nerve without direct physical contact.

Optical parameter variability in laser nerve stimulation: a study of pulse duration, repetition rate, and wavelength.

Pulsed lasers can evoke neural activity from motor as well as sensory neurons in vivo. Lasers allow more selective spatial resolution of stimulation than the conventional electrical stimulation. To date, few studies have examined pulsed, mid-infrared laser stimulation of nerves and very little of the available optical parameter space has been studied. In this study, a pulsed diode laser, with wavelength between 1.844-1.873 microm, was used to elicit compound action potentials (CAPs) from the auditory system of the gerbil. We found that pulse durations as short as 35 micros elicit a CAP from the cochlea. In addition, repetition rates up to 13 Hz can continually stimulate cochlear spiral ganglion cells for extended periods of time. Varying the wavelength and, therefore, the optical penetration depth, allowed different populations of neurons to be stimulated. The technology of optical stimulation could significantly improve cochlear implants, which are hampered by a lack of spatial selectivity.

Selectivity of neural stimulation in the auditory system: a comparison of optic and electric stimuli.

Pulsed, mid-infrared lasers were recently investigated as a method to stimulate neural activity. There are significant benefits of optically stimulating nerves over electrically stimulating, in particular the application of more spatially confined neural stimulation. We report results from experiments in which the gerbil auditory system was stimulated by optical radiation, acoustic tones, or electric current. Immunohistochemical staining for the protein c-FOS revealed the spread of excitation. We demonstrate a spatially selective activation of neurons using a laser; only neurons in the direct optical path are stimulated. This pattern of c-FOS labeling is in contrast to that after electrical stimulation. Electrical stimulation leads to a large, more spatially extended population of labeled, activated neurons. In the auditory system, optical stimulation of nerves could have a significant impact on the performance of cochlear implants, which can be limited by the electric current spread.

In vivo glucose measurement by surface-enhanced Raman spectroscopy.

This paper presents the first in vivo application of surface-enhanced Raman scattering (SERS). SERS was used to obtain quantitative in vivo glucose measurements from an animal model. Silver film over nanosphere surfaces were functionalized with a two-component self-assembled monolayer, and subcutaneously implanted in a Sprague-Dawley rat such that the glucose concentration of the interstitial fluid could be measured by spectroscopically addressing the sensor through an optical window. The sensor had relatively low error (RMSEC = 7.46 mg/dL (0.41 mM) and RMSEP = 53.42 mg/dL (2.97 mM).

Laser stimulation of the auditory nerve.

BACKGROUND AND OBJECTIVES: For centuries, electric current has been used to stimulate neurons. Shortcomings of electrical stimulation include the contact between the stimulating electrode and the tissue, and the non-selective stimulation of the tissue. In contrast to electric stimulation, optical radiation can provide spatially selective neural stimulation without tissue contact. STUDY DESIGN/MATERIALS AND METHODS: Acute in vivo experiments using gerbils were conducted to record optically evoked compound action potentials (CAPs) from the cochlea. RESULTS: Optical radiation evokes CAPs in normal hearing animals and in deafened animals, in which cochleae lack outer and inner hair cells. Stimulation threshold was measured as 0.018+/-0.003 J/cm(2) (mean+/-SE). Laser radiation could be increased by 30-40 dB until drastic changes were seen in cochlear function. Cochlear response amplitudes to optical radiation were stable over extended stimulation times. CONCLUSIONS: We have demonstrated that the auditory nerve can be stimulated by optical radiation. One potential clinical use of this technology would be for cochlear implants.

Stokes polarimetry imaging of rat tail tissue in a turbid medium: degree of linear polarization image maps using incident linearly polarized light.

Illumination with incident linearly polarized light on tissue and polarization state measurements of the remitted light provide a means by which various tissue structures can be differentiated. A rat tail is embedded within a turbid gelatin such that there is a variable depth of medium above it. By varying the incident polarization angle (IPA) of the illuminating linearly polarized light, the geometry, and the orientation angle of the tissue, a series of 2-D degree of linear polarization image maps are created using our Stokes polarimetry imaging technique. The image maps show locations of the polarization-sensitive structures in the rat tail, including soft tissue, intervertebral disks, and tendons. The observed morphologies in the image maps indicate locations where the depolarization of light differs according to the tissue type and underlying layers. The data indicate the importance of varying the IPA, and that tissue dichroism and birefringence affect the degree of linear polarization image maps. Diagnostic information regarding subsurface tissue structures is obtained.

Stokes polarimetry imaging of rat-tail tissue in a turbid medium using incident circularly polarized light.

BACKGROUND AND OBJECTIVES: We describe a Stokes polarimetry imaging technique that quantifies the polarization properties of remitted light backscattered from a sample. STUDY DESIGN/MATERIALS AND METHODS: Right- and left-circularly polarized near-infrared light was used to illuminate rat-tail tissue embedded in turbid gelatin. RESULTS: The degree of linear polarization (DoLP) and degree of circular polarization (DoCP) image-maps indicate that increasing the depth of the rat tail within the turbid medium and varying the rat-tail geometry and orientation relative to the light source affected the contrast between structures and adjacent tissue layers. CONCLUSION: Stokes polarimetry imaging shows that the intervertebral discs and soft tissue regions of rat tails strongly depolarize incident circularly polarized light. Tendon regions remit light with a more linear form due to birefringence. Both DoLP and DoCP image-maps provide contrast between tissue structures. When differentiating between unpolarized light and light with low DoCP or DoLP, the polarization of backscattered light from the turbid medium must to be taken into consideration.

Real-time glucose sensing by surface-enhanced Raman spectroscopy in bovine plasma facilitated by a mixed decanethiol/mercaptohexanol partition layer.

A new, mixed decanethiol (DT)/mercaptohexanol (MH) partition layer with dramatically improved properties has been developed for glucose sensing by surface-enhanced Raman spectroscopy. This work represents significant progress toward our long-term goal of a minimally invasive, continuous, reusable glucose sensor. The DT/MH-functionalized surface has greater temporal stability, demonstrates rapid, reversible partitioning and departitioning, and is simpler to control compared to the tri(ethylene glycol) monolayer used previously. The data herein show that this DT/MH-functionalized surface is stable for at least 10 days in bovine plasma. Reversibility is demonstrated by exposing the sensor alternately to 0 and 100 mM aqueous glucose solutions (pH approximately 7). The difference spectra show that complete partitioning and departitioning occur. Furthermore, physiological levels of glucose in two complex media were quantified using multivariate analysis. In the first system, the sensor is exposed to a solution consisting of water with 1 mM lactate and 2.5 mM urea. The root-mean-squared error of prediction (RMSEP) is 92.17 mg/dL (5.12 mM) with 87% of the validation points falling within the A and B range of the Clarke error grid. In the second, more complex system, glucose is measured in the presence of bovine plasma. The RMSEP is 83.16 mg/dL (4.62 mM) with 85% of the validation points falling within the A and B range of the Clarke error grid. Finally, to evaluate the real-time response of the sensor, the 1/e time constant for glucose partitioning and departitioning in the bovine plasma environment was calculated. The time constant is 28 s for partitioning and 25 s for departitioning, indicating the rapid interaction between the SAM and glucose that is essential for continuous sensing.