Direct Measurement of a Biomarker's Native Optimal Frequency with Physical Adsorption Based Immobilization.

The optimal frequency (OF) of a biomarker in electrochemical impedance spectroscopy (EIS) is the frequency at which the EIS response best reflects the binding of the biomarker to its molecular recognition element. Commonly, biosensors rely on complicated immobilization chemistry to attach biological molecules to the sensor surface, making the direct study of a biomarker's native OF a challenge. Physical adsorption presents a simple immobilization strategy to study the native biomarker's OF, but its utility is often discouraged due to a loss in biological activity. To directly study a biomarker's native OF and investigate the potential of OF to overcome the limitations of physical adsorption, a combination of EIS and glutaraldehyde-mediated physical adsorption was explored. The experimental sensing platform was prepared by immobilizing either anti-lactoferrin (Lfn) IgG or anti-immunoglobulin E (IgE) onto screen printed carbon electrodes. After characterizing the native OFs of both biomarkers, investigation of the platform's specificity, stability, and performance in complex medium was found to be sufficient. Finally, a paper-based tear sampling component was integrated to transform the testing platform into a prototypical point-of-care dry eye diagnostic. The investigation of native OFs revealed a correlation between the native OFs (57.44 and 371.1 Hz for Lfn and IgE, respectively) and the molecular weight of the antibody-antigen complex. Impedance responses at the native OFs have enabled detection limits of 0.05 mg/mL and 40 ng/mL for Lfn and IgE, respectively, covering the clinically relevant ranges. The native OFs were found to be robust across various testing mediums and conditions.

Novel fungal FAD glucose dehydrogenase derived from Aspergillus niger for glucose enzyme sensor strips.

In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases.

Detection of 1,5-Anhydroglucitol by Electrochemical Impedance Spectroscopy.

Multiple markers are used to assess glycemic control in patients with diabetes mellitus (DM). New technology that permits simultaneous detection of multiple biomarkers combined with those used at the point of care indicative of glycemic control, including glycemic variability determined from 1,5-anhydroglucitol measurement, could provide better management and further insight into the disease. This platform was based on previous research involving glucose detection and uses electrochemical impedance spectroscopy to detect a range of 1,5-anhydroglucitol concentrations at an optimal binding frequency. The enzyme pyranose oxidase was fixed to gold electrodes while a sine wave of sweeping frequencies was induced in purified solutions and in variable presence of whole blood. The optimal binding frequency for the detection of 1,5-anhydroglucitol was found to be 3.71 kHz. The impedance response compared to the concentration of target present was found to have a logarithmic slope of 7.04 with an R-squared value of 0.96. This response includes 2 experimental sets, a single test of a low concentration range and a high concentration range with 5 replicates. The relative standard deviation of the high range varied from 28% to 27% from lowest to highest concentrations. Best detection in complex solutions was found in lower blood concentrations of 0.5% and 1%, but maintained relatively high accuracy in concentrations 5% and 10%. The sensor platform was successfully evaluated at a high dynamic range of 1,5-AG in purified solutions. In the presence of whole blood, lowest percentages yielded the best results indicating that filtering interferents may be necessary in final device architecture.

The promise of electrochemical impedance spectroscopy as novel technology for the management of patients with diabetes mellitus.

Self-monitoring of blood glucose is the standard of care in management of hyperglycemia among patients with diabetes mellitus. To increase the sensitivity and specificity of current devices, a novel method of detecting glucose using electrochemical impedance spectroscopy (EIS) technology is explored. The enzyme glucose oxidase (GOx) was fixed to gold electrodes and a sine wave of sweeping frequencies was induced using a wide range of concentrations of glucose. Each frequency in the impedance sweep was analyzed for the highest response and R-squared value. The frequency with both factors optimized is specific for the glucose-GOx binding interaction and was determined to be 1.17 kHz in purified solutions in both higher and lower ranges of glucose. The correlation between the impedance response and concentration at the low range of detection (0-100 mg dL(-1) of glucose) was determined to be 3.53 ohm/ln (mg dL(-1)) with an R-squared value of 0.90 with a 39 mg dL(-1) lower limit of detection. The same frequency of 1.17 kHz was verified in whole blood under the same glucose range. The above data confirm that EIS offers a new method of glucose detection as an alternative to current technology in use by patients. Additionally, the unique frequency response of individual markers allows for modulation of signals so that several other markers important in the management of diabetes could be measured with a single sensor.

Immobilization of functional light antenna structures derived from the filamentous green bacterium Chloroflexus aurantiacus.

The integration of highly efficient, natural photosynthetic light antenna structures into engineered systems while their biophotonic capabilities are maintained has been an elusive goal in the design of biohybrid photonic devices. In this study, we report a novel technique to covalently immobilize nanoscaled bacterial light antenna structures known as chlorosomes from Chloroflexus aurantiacus on both conductive and nonconductive glass while their energy transducing functionality was maintained. Chlorosomes without their reaction centers (RCs) were covalently immobilized on 3-aminoproyltriethoxysilane (APTES) treated surfaces using a glutaraldehyde linker. AFM techniques verified that the chlorosomes maintained their native ellipsoidal ultrastructure upon immobilization. Results from absorbance and fluorescence spectral analysis (where the Stokes shift to 808/810 nm was observed upon 470 nm blue light excitation) in conjunction with confocal microscopy confirm that the functional integrity of immobilized chlorosomes was also preserved. In addition, experiments with electrochemical impedance spectroscopy (EIS) suggested that the presence of chlorosomes in the electrical double layer of the electrode enhanced the electron transfer capacity of the electrochemical cell. Further, chronoamperometric studies suggested that the reduced form of the Bchl- c pigments found within the chlorosome modulate the conduction properties of the electrochemical cell, where the oxidized form of Bchl- c pigments impeded any current transduction at a bias of 0.4 V within the electrochemical cell. The results therefore demonstrate that the intact chlorosomes can be successfully immobilized while their biophotonic transduction capabilities are preserved through the immobilization process. These findings indicate that it is feasible to design biophotonic devices incorporating fully functional light antenna structures, which may offer significant performance enhancements to current silicon-based photonic devices for diverse technological applications ranging from CCD devices used in retinal implants to terrestrial and space fuel cell applications.

Characterization of Chlorobium tepidum chlorosomes: a calculation of bacteriochlorophyll c per chlorosome and oligomer modeling.

The bacteriochlorophyll (Bchl) c content and organization was determined for Chlorobium (Cb.) tepidum chlorosomes, the light-harvesting complexes from green photosynthetic bacteria, using fluorescence correlation spectroscopy and atomic force microscopy. Single-chlorosome fluorescence data was analyzed in terms of the correlation of the fluorescence intensity with time. Using this technique, known as fluorescence correlation spectroscopy, chlorosomes were shown to have a hydrodynamic radius (Rh) of 25 +/- 3.2 nm. This technique was also used to determine the concentration of chlorosomes in a sample, and pigment extraction and quantitation was used to determine the molar concentration of Bchl c present. From these data, a number of approximately 215,000 +/- 80,000 Bchl c per chlorosome was determined. Homogeneity of the sample was further characterized by dynamic light scattering, giving a single population of particles with a hydrodynamic radius of 26.8 +/- 3.7 nm in the sample. Tapping-mode atomic force microscopy (TMAFM) was used to determine the x,y,z dimensions of chlorosomes present in the sample. The results of the TMAFM studies indicated that the average chlorosome dimensions for Cb. tepidum was 174 +/- 8.3 x 91.4 +/- 7.7 x 10.9 +/- 2.71 nm and an overall average volume 90,800 nm(3) for the chlorosomes was determined. The data collected from these experiments as well as a model for Bchl c aggregate dimensions was used to determine possible arrangements of Bchl c oligomers in the chlorosomes. The results obtained in this study have significant implications on chlorosome structure and architecture, and will allow a more thorough investigation of the energetics of photosynthetic light harvesting in green bacteria.