Cortisol, a biomarker of stress, has recently been shown to have potential in evaluating the physiological state of individuals diagnosed with stress-related conditions including chronic fatigue syndrome. Noninvasive techniques to extract biomarkers from the body are a topic of considerable interest. One such technique to achieve this is known as reverse iontophoresis (RI) which is capable of extracting biomolecules through the skin. Unfortunately, however, the extracted levels are often considerably lower in concentration than those found in blood, thereby requiring a very sensitive analytical method with a low limit of detection. A promising sensing approach, which is well suited to handle such samples, is Surface Plasmon Resonance (SPR) spectroscopy. When coupled with aptamer modified surfaces, such sensors can achieve both selectivity and the required sensitivity. In this study, fabrication and characterization of a RIbased SPR biosensor for the measurement of cortisol has been developed. The optical mount and diffusion cell were both fabricated through the use of 3D printing techniques. The SPR sensor was configured to employ a prism couplerbased arrangement with a laser generation module and CCD line sensor. Cortisol-specific DNA aptamers were immobilized onto a gold surface to achieve the necessary selectivity. For demonstration purposes, cortisol was extracted by the RI system using a skin phantom flow system capable of generating time dependent concentration profiles. The captured sample was then transported using a micro-fluidic platform from the RI collection site to the SPR sensor for real-time monitoring. Analysis and system control was accomplished within a developed LabVIEW® program.
Recent developments in the identification of biomarkers offer a potential means to facilitate early disease detection, gauge treatment in drug therapy clinical trials, and to assess the impact of fatigue and/or stress as related to human physical and cognitive performance. For practical implementation, however, real-time sensing and quantification of such physiological biomarkers is preferred. Some key aspects in this process are continuous sample collection and real time detection. Traditionally, blood is considered the gold standard for samples but frequent phlebotomy is painful and inconvenient. Other sources like saliva and passive sweat cannot be precisely controlled and are affected by other limitations. Some of these can be addressed by reverse iontophoresis which is a noninvasive technique capable of facilitating controlled transport of biomolecules up to 20kDa in size across the skin barrier by passing a low level current between two dermal electrodes. The samples collected at the electrode site can then be monitored at site or transported via a microfluidic channel towards a sensor. In the case reported here, the sensor is based on surface plasmon resonance (SPR), which is a label free, real time, and highly sensitive optical sensing technique. The real time SPR detection of targeted biomarkers is then achieved through the use of aptamer surface modification. In this experiment, extraction and detection of orexin A, a stress related biomarker, is used for demonstration purposes.
In the past, Faraday based optical polarimetry approaches have shown considerable potential for the measurement of optical activity with application towards the noninvasive measurement of physiological glucose concentration. To date, most reported closed-loop systems incorporate separate Faraday components for modulation and compensation requiring two optical crystals. These systems have demonstrated significant stability and sub-millidegree rotational sensitivities; however, the main drawbacks to this approach are the optical materials (e.g., terbium gallium garnet) can be quite expensive and often custom fabricated induction coils are required. In this investigation, we propose a new design for the Faraday components capable of achieving both modulation and compensation in a single crystal device. The design is more compact and is capable of achieving similar performance with low cost commercially available inductive components. To facilitate prototype optimization, our group has developed a finite element model (FEM) that can simulate various physical parameters such as geometry, inductance, and orientation with respect to the optical rod in order to minimize power consumption and size while maintaining appropriate field strength. Performance is comparable to existing nonintegrated approaches and is capable of achieving modulation depths < 1° under similar operating conditions while attaining sub-millidegree linear polarization sensitivity. There is also excellent correlation between the FEM and experimental prototype with operational performance shown to be within 1.8%. The use of FEM simulations allows for the analysis of a vast range of parameters before prototypes are fabricated and can facilitate custom designs as related to development time, anticipated performance, and cost reduction.
In recent years, significant advances have been made in the development of noninvasive polarimetric glucose detection systems, salutary for the treatment of our rapidly increasing diabetic population. This area of research utilizes the aqueous humor as the detection medium for its strong correlation to blood glucose concentration and highlights three major features: the optical activity of glucose, minimal scattering of the medium, and the ability to detect sub-millidegree rotation in polarized light. However, many of the current polarimetric systems are faced with size constraints based on the paramount optical components. As a step toward developing a low cost hand-held design, our group has designed a miniaturized integrated single-crystal Faraday modulator/compensator. This device is capable of replacing the traditional two component arrangement that has been widely reported on in many Faraday-based polarimetric configurations. In this study, the newly designed prototype is compared with a theoretical model and its performance is evaluated experimentally under both noninvasive static and dynamic glucose monitoring conditions. The combined rotator can achieve modulation depths above 1°, and when operating in a compensated closed-loop configuration, it has demonstrated glucose prediction errors of 1.8 mg/dL and 5.4 mg/dL under hypoglycemic and hyperglycemic conditions, respectively. These results demonstrate that such an integrated design can perform similar if not better than its larger two-part predecessors. This technology could also be extended to facilitate the use of multispectral polarimetry by considerably reducing the required number of physical components. Such multispectral techniques have demonstrated usefulness for in vivo and multi-analyte noninvasive sensing.
The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic
measure in diabetes to determine a history of glycemic compliance. Depending on a protein’s half-life in blood,
compliance can be assessed from a few days to several months in the past, which can then be used to provide additional
therapeutic guidance. Current glycated protein detection methods are limited in their ability to measure multiple proteins,
and are susceptible to interference from other blood pathologies. In this study, we developed and characterized DNA
aptamers for use in Surface Plasmon Resonance (SPR) sensors to assess the blood protein hemoglobin. The aptamers
were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process
which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this
process are sequenced and identified aptamers are then synthesized. The SELEX process was performed to produce
aptamers for a glycated form of hemoglobin. Equilibrium dissociation constants for the binding of the identified aptamer
to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitted Langmuir isotherms obtained through
SPR. These constants were determined to be 94 nM, 147 nM, and 244 nM respectively. This aptamer can potentially be
used to create a SPR aptamer based biosensor for detection of glycated hemoglobin, a technology that has the potential to
deliver low-cost and immediate glycemic compliance assessment in either a clinical or home setting.
Molecularly imprinted polymer (MIP) thin films and surface plasmon resonance (SPR) sensing technologies were
combined to develop a novel sensing platform for monitoring real-time theophylline concentration, which is a compound
of interest in environmental monitoring and a molecular probe for phenotyping certain cytochrome P450 enzymes. The
MIPs hydrogel is easy to synthesize and provides shape-selective recognition with high affinity to specific target
molecules. Different polymerization formulas were tested and optimized. The influence of the monomer sensitive factors
were addressed by SPR. SPR is an evanescent wave optics based sensing technique that is suitable for real-time and label
free sensing purposes. Gold nanorods (Au NRs) were uniformly immobilized onto a SPR sensing surface for the
construction of a fiber optics based prism-free localized SPR (LSPR) measurement. This technique can be also applied to
assess the activities of other small organic molecules by adjusting the polymerization formula, thus, this approach also
has many other potential applications.
The peak extinction wavelength of the nano-size noble metal localized surface plasmon resonance (LSPR) spectrum is
unexpectedly sensitive to nanoparticle size, shape, and local external dielectric environment. This sensitivity to the
environment has enabled the development of a new class of nanoscale affinity biosensors. Aptamer (single strand DNA)
based gold nanorods (Au NRs) and magnetic beads (MBs) combined LSPR biosensor has been developed for the rapid
and label-free detection of glycated proteins in small solution volumes. An aptamer self-assembly monolayer (SAM)
functionalized surface plasmon resonance sensor has also been developed for comparison purposes. For demoonstration
purposes, albumin and thrombin are used initially as the target proteins. The ability to monitor such molecules in the
body could facilitate the diagnosis and treatment of diabetic patients.
There is a need to effectively and accurately monitor physiological glucose levels in individuals afflicted
with diabetes mellitus. One promising noninvasive technique involves the use of optical polarimetry, in which the
eye is commonly used as the sensing location. Since glucose is a chiral molecule, it has the ability to rotate plane
polarized light by an amount that is proportional to glucose concentration. It has also been shown that glucose levels
in the aqueous humor of the eye correlate well to those of blood. Therefore, we will report on an in vivo study that
is conducted using a New Zealand White (NZW) rabbit model in conjunction with a custom developed Faradaybased
optical polarimeter with sub-millidegree resolution. All animals used in this investigation were anesthetized
with isoflurane and an insulin/dextrose protocol was used to control blood glucose concentration. A polarized laser
light (632.8nm HeNe) signal was coupled through the anterior chamber of the eye using a custom designed ocular
apparatus. System calibration was performed through measurement of the detected optical polarimetric signal and
corresponding discrete blood glucose measurements taken with a handheld glucometer. Reference blood glucose
samples were also measured using a YSI 2300+ glucose analyzer. The study results show that physiological glucose
can be predicted with error levels on the order of 15%.
Physiological glucose monitoring is important aspect in the treatment of individuals afflicted with diabetes
mellitus. Although invasive techniques for glucose monitoring are widely available, it would be very beneficial to
make such measurements in a noninvasive manner. In this study, a New Zealand White (NZW) rabbit animal model was utilized to evaluate a developed iris-based imaging technique for the in vivo measurement of physiological glucose concentration. The animals were anesthetized with isoflurane and an insulin/dextrose protocol was used to control blood glucose concentration. To further help restrict eye movement, a developed ocular fixation device was used. During the experimental time frame, near infrared illuminated iris images were acquired along with corresponding discrete blood glucose measurements taken with a handheld glucometer. Calibration was performed using an image based Partial Least Squares (PLS) technique. Independent validation was also performed to assess model performance along with Clarke Error Grid Analysis (CEGA). Initial validation results were promising and show that a high percentage of the predicted glucose concentrations are within 20% of the reference values.
A potential noninvasive glucose sensing technique was investigated for application towards in vivo
glucose monitoring for individuals afflicted with diabetes mellitus. Three dimensional ray tracing simulations
using a realistic iris pattern integrated into an advanced human eye model are reported for physiological
glucose concentrations ranging between 0 to 500 mg/dL. The anterior chamber of the human eye contains a
clear fluid known as the aqueous humor. The optical refractive index of the aqueous humor varies on the
order of 1.5x10-4 for a change in glucose concentration of 100 mg/dL. The simulation data was analyzed with
a developed multivariate chemometrics procedure that utilizes iris-based images to form a calibration model.
Results from these simulations show considerable potential for use of the developed method in the prediction
of glucose. For further demonstration, an in vitro eye model was developed to validate the computer based
modeling technique. In these experiments, a realistic iris pattern was placed in an analog eye model in which
the glucose concentration within the fluid representing the aqueous humor was varied. A series of high
resolution digital images were acquired using an optical imaging system. These images were then used to
form an in vitro calibration model utilizing the same multivariate chemometric technique demonstrated in the
3-D optical simulations. In general, the developed method exhibits considerable applicability towards its use
as an in vivo platform for the noninvasive monitoring of physiological glucose concentration.
The objective of this study was to develop rapid, inexpensive, and easily applied in vivo phenotyping strategies for
characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP) enzymes in biological
fluids. Therefore, the accurate detection of low concentration of theophylline, which can be used as a probe for
cytochrome P450 (CYP450) enzymes (e.g. CYP1A2) activity, could benefit drug-metabolizing studies. In this study, a
portable, specific, and sensitive functionalized surface plasmon resonance (SPR) sensor using polyacrylamide
molecularly imprinted polymers (MIPs) as the highly specific selector is developed for the detection of low
concentration theophylline in the presence of other confounding components, such as, caffeine which has a very similar
chemical structure.
A need exists for the continued development of diagnostic tools and methods capable of distinguishing and characterizing slight differences in the optical properties of tissues. We present a method to estimate the scattering coefficient contribution as a function of particle size in complex mixtures of polystyrene spheres. The experimental method we used is a Mueller matrix imaging approach. The Mueller matrix encodes the polarization-dependent properties of the sample and describes how a given sample will transform an incident light polarization state. A partial least-squares approach is used to form a model around a set of Mueller matrix image-based measurements to accurately predict the individual scattering coefficient contributions in phantoms containing 0.2, 0.5, 1, and 2 µm-diameter polystyrene spheres. The results show individual scattering coefficient contribution errors as low as 0.1585 cm–1 can be achieved. In addition, it is shown how the scattering type (i.e., Rayleigh and Mie) is encoded within the Mueller matrix. Such methods may eventually lead to the development of improved diagnostic tools capable of characterizing and distinguishing between tissue abnormalities, such as superficial cancerous lesions from their benign counterparts.
Recently, polarization based optical approaches have received considerable interest due to their potential medical applications. Glucose, a chiral molecule, has the ability to rotate the plane of linearly polarized light, commonly referred to as optical activity, as well as affecting the refractive index of the media which is therefore affects the overall scattering coefficient in a given media. The magnitude of each effect is related to the concentration of glucose. Based on these effects, it would be expected that a change in glucose concentration would alter the diffuse reflectance polarization patterns from turbid media. In this study, we investigate how each of these effects is correlated to glucose concentration in a physiological range for highly scattering biological media. Furthermore, it is shown how diffusely polarized imaging when coupled with chemometrics techniques can be used to quantify glucose concentration.
The development of a rapid, inexpensive, and accurate in vivo phenotyping methodology for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP450) enzymes would be very beneficial. In terms of application, in the wake of the human genome project, considerable interest is focused on the development of new drugs whose uses will be tailored to specific genetic polymorphisms, and on the individualization of dosing regimens that are also tailored to meet individual patient needs depending upon genotype. In this investigation, chemical probes for CYP450 enzymes were characterized and identified with Raman spectroscopy. Furthermore, gold-based metal colloid clusters were utilized to generate surface enhanced Raman spectra for each of the chemical probes. Results will be presented demonstrating the ability of SERS to identify minute quantities of these probes on the order needed for in vivo application.
In this paper we present experimental results demonstrating processing techniques developed in our laboratory that can be utilized to decode or extract useful information from two-dimensional Mueller matrices of turbid media. Through the use of these methods, involving the partial least squares technique, it is shown how scattering coefficient contributions as a function of particle size can be estimated for a given sample. Furthermore, we demonstrate how a spatial selection algorithm known as "chain select" can be used to help facilitate the interpretation of the measured Mueller matrix images. The samples utilized in this investigation were comprised of polystyrene spheres with diameters ranging from 200 nm to 2000 nm and analyzed with 514 nm light. At this wavelength, both Rayleigh and Mie-types of scattering are observed.
In the recent past, optical polarimetry has been shown as a potential method for noninvasive physiologic glucose sensing in the eye. Although the necessary sensitivity and accuracy have been demonstrated experimentally through in vitro studies using a range of media from simplistic glucose doped-water to more complex media such as aqueous humor, the main problem currently hindering long-term in vivo measurements is corneal birefringence coupled with motion artifact. This is due to the inability to distinguish E-field rotation due to glucose from the effects of time varying corneal birefringence. In this investigation, the effect of corneal birefringence will be discussed and a potential method to overcome this problem will be presented with supporting results.
In this investigation, a polarization-based imaging system is developed and described that measures the two-dimensional effective backscattering Mueller matrix of a sample in near real-time. As is well known, a Mueller matrix can provide considerable information on the makeup and optical characteristics of a sample and also directly describes how the sample transforms an incident light beam. The ability to measure the two-dimensional Mueller matrix of a biological sample, therefore, can provide considerable information on the sample composition as well as the potential to reveal significant structural information that normally would not be visible through standard imaging techniques. Additional information can also be obtained through the application of image-processing, decomposition, and reconstruction techniques that operate directly on the 2D Mueller matrix. Using the developed system, it is shown how the induction of internal strain within the sample coupled with image reconstruction and decomposition techniques can further improve image contrast and aid in the detection of boundaries between tissues of different biomechanical and structural properties. The studies presented were performed with both rat tissue and a melanoma-based tissue culture. The results demonstrate how these techniques could provide information that may be of diagnostic value in the physical detection of malignant lesion boundaries.
Optical Coherence Tomography (OCT) is a relatively new type of imaging system for medical diagnosis. Because most current OCT systems use a sharply focused beam in tissues, they have a short depth of field (high image resolution is near the focus only). In this paper, limited diffraction beams of different orders are used to increase depth of field and to reduce sidelobes in OCT. Results show that the proposed OCT system has a lateral resolution of about 4.4 wavelengths (the central wavelength of the source is about 940 nm with a bandwidth of about 70 nm) and lower than -60 dB sidelobes over an entire depth of field of 4.5 mm with the diameter of the objective lens of 1 mm.
Optical Coherence Tomography (OCT) is a relatively new type of imaging system for medical diagnosis. Because most current OCT systems use a sharply focused beam in tissues, they have a short depth of field (high image resolution is near the focus only). In this paper, limited diffraction beams of different orders are used to increase depth of field and to reduce sidelobes in OCT. Results show that the proposed OCT system has a lateral resolution of about 4.4 wavelengths (the central wavelength of the source is about 940 nm with a bandwidth of about 70 nm) and lower than -60 dB sidelobes over an entire depth of field of 4.5 mm with the diameter of the objective lens of 1 mm.
The application of optical polarimetry, using the anterior chamber of the eye as the sensing site, is being investigated as a potential method to develop a noninvasive physiological glucose monitor. First, we present results characterizing the optical rotatory dispersion of the main optically active analytes found within the aqueous humor of the eye including, glucose, albumin, and ascorbic acid. This information is used in conjunction with multiple linear regression to demonstrate how multispectral polarimetry can be used to minimize glucose prediction error in samples containing varying physiological concentrations of glucose and albumin. For this multispectral study, a novel dual wavelength (532 nm and 635 nm) polarimeter was designed and constructed. This sensor is novel in that it provides simultaneous measurements using a 532 nm laser in an open- loop configuration and a 635 nm laser in a closed-loop configuration. In addition, we present in vivo results using New Zealand White rabbits that indicate the time delay between blood and aqueous human glucose levels is below ten minutes. Lastly, we provide preliminary in vivo polarimetric results and discuss the main issues currently hindering the measurement of glucose.
Wavelength selection is an important preprocessing step for improving and simplifying calibrations in both quantitative and qualitative problems. An improved variable selection algorithm has been developed to improve upon existing methods in terms of speed and prediction error. The new technique uses a novel peak-hopping strategy to move quickly between important spectral regions. Results when applied to both Raman and near-infrared data show that the algorithm is very fast, decreases prediction errors, and chooses a small subset of the full spectral range available. A comparison with other techniques is given and the respective advantages and disadvantages are discussed.
In our investigation, we present both multi-spectral in vitro and preliminary single wavelength in vivo results supporting the use of optical polarimetry as a potential non-invasive method for glucose sensing. The site utilized for our in vivo measurements is the anterior chamber of the eye in a rabbit model. The anterior chamber of the eye contains a relatively clear and minimal scattering fluid known as the aqueous humor. The glucose levels of the aqueous humor are correlated to those of blood, therefore providing a mechanism to indirectly estimate blood glucose levels. A device to effectively couple light through the anterior chamber is also presented. As for the in vitro experiments, a multi-spectral approach is demonstrated as a method to minimize prediction error when glucose is not the only optically active component that varies in concentration.
In this preliminary investigation, a two wavelength optical polarimetric system was used to show the potential of the approach to be used as an in vivo noninvasive glucose monitor. The dual wavelength method is shown as a means of overcoming two of them ore important problems with this approach for glucose monitoring, namely, motion artifact and the presence of other optically chiral components. The use of polarized light is based on the fact that the polarization vector of the light rotates when it interacts with an optically active material such as glucose. The amount of rotation of the light polarization is directly proportional to the optically active molecular concentration and to the sample path length. The end application of this system would be to estimate blood glucose concentrations indirectly by measuring the amount of rotation of the light beam's polarization state due to glucose variations within the aqueous humor of the anterior chamber of the eye. The system was evaluated in vitro in the presence of motion artifact and in combination with albumin, another interfering optical rotatory chemical component. It was shown that the dual wavelength approach has potential for overcoming these problems.
In order to enhance cell culture growth in bioreactors, biosensors such as those used for glucose detection must be developed that are capable of monitoring cell culture processes continuously and preferably noninvasively. The development of a unique noninvasive, optically based polarimetric glucose sensor is reported. The data were collected using a highly sensitive, lab-built polarimeter with digital feedback and a red laser diode source. A range of glucose concentrations was evaluated using both glucose-doped double-distilled water and a bovine serum-based medium. The serum-based medium is the nutritional environment in which the cell cultures are grown. Both media were examined across two glucose concentration ranges—a lower range of 100 mg/dl in 10-mg/dl increments and a higher range up to 600 mg/dl in 50-mg/dl increments. The linear regression in all experiments yielded standard errors of prediction of less than 8.5 mg/dl across both ranges.
The use of polarimetry in the investigation of chiral molecules has been researched for over a century. However, it has not been until recently that the sensitivity and accuracy of this technology has improved enough to be applied to the quantification of the low optically metabolite concentrations seen within the body. The long term goal of this research is the development of a polarimetric detection system with presence of other confounders. In this study, a robust polarimeter utilizing digital closed-loop control was designed and constructed that can effectively measure millidegree rotations of plane- polarized light within biological media. In vitro experiments were conducted using a 1 cm path length sample cell in both glucose doped cell culture and bovine aqueous humor media with analyte concentrations on the order of those seen in the body. A high degree of linearity between the measured signal and glucose concentration is seen during calibration of both the cell culture and aqueous humor experiments with correlation coefficients of 0.9995 and 0.9912, respectively. In addition, validation of the obtained calibration models yielded standard errors of prediction of 8.469 and 20.25 mg/dl for each media, respectively. Overall, we feel the conducted experiments are a logical step to furthering the development of using polarimetry in the detection of optically active metabolites, and our results indicate that accurate detection of glucose in the presence of additional confounders can be accomplished in both cell culture and aqueous humor media.
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