Kerry P. McNamara and Zeev Rosenzweig*
Department of Chemistry
University of New Orleans, New Orleans, LA 70148
The development of glucose sensors displaying high sensitivity, fast response, reproducibility and long term stability has been a main target in sensor research during the last three decades. Much effort is devoted to the development of new and improved amperometric glucose sensors that will continuously monitor physiological levels of glucose in blood over an extended period of time (1-5). Another direction in recent sensor research is the development of submicrometer sized sensors.
Miniaturized glucose sensors are particularly attractive in connection with various ex-vivo and in-vivo clinical applications including measurements of glucose in extremely small volumes or monitoring of localized events where a spatial resolution of 10 micrometer is desired.
Traditionally the method of choice is the design of glucose microelectrodes (6,7)
Two alternative optochemical approaches for glucose sensing in small volumes are described in this manuscript:
Micrometer sized fiber optic glucose biosensor - The small size of this sensor is realized by growing a glucose sensitive polymer at the end of a pulled optical fiber tip (see figure 1). The sensor is based on the enzymatic reaction of glucose oxidase which catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide while consuming oxygen.
Tris (1,10 phenanthroline) ruthenium chloride, an oxygen indicator is used as a transducer. It displays a strong absorption in the visible region (lmax = 447 nm, e = 18100) a strong fluorescence (lmax = 604 nm, corrected), a high emission quantum efficiency of 0.4, a relatively long excited state lifetime (around 1 msec), and a high photostability. These luminescence properties result in a high Stern-Volmer quenching constant and an efficient quenching by molecular oxygen. The ruthenium complex and glucose oxidase are incorporated into acrylamide polymer that is attached covalently to a silanized optical fiber tip surface by photo-initiated polymerization. An inverted Olympus microscope based optical fiber sensor setup is described in figure 2. An argon ion (American) laser at 488 nm is used as a light source. A high precision single mode optical fiber coupler (Newport) is used to direct the laser beam into the fiber sensor tip. The sensor reagent is excited by the incident beam and the fluorescence light as well as the excitation light are collected by a 20 X objective lens. A 3 mirrors 150 mm spectrograph (Acton) with 300 grooves/mm grating is used to disperse the fluorescence signal. A high performance charge coupled device camera (Princeton instruments) is used for spectral data collection. A compatible PC microcomputer is used for data acquisition and analysis. Due to its small size and the lack of membrane support the response time of the sensor is only 2 seconds. An absolute detection limit of around 1 x 10-15 mol is achieved. The new glucose sensor is at least 25 times faster and its absolute sensitivity is 5-6 orders of magnitude higher than that of current glucose sensors. The advantages of fiber optic fluorescence sensors over electrochemical sensors include miniaturization, geometric flexibility and the lack of electrical connection between the sensor and the sample.
Glucose Measurement using Liposome-Based Optochemical Oxygen Sensors - Submicrometer sized optochemical sensors offer significant improvements in their absolute detection limit, response time, and spatial resolution compared to conventional micrometer or millimeter sized chemical sensors and biosensors (8-11). However, the use of an optical fiber or a micropipette as a support, limits the applicability of the method in biological research, particularly in the field of single cell analysis, where the sensor has to be inserted into a single cell through the cell membrane. While the measurement technique is not as invasive as conventional cellular fluorescence labeling (12), the damage to the cell membrane may affect cellular properties. Furthermore, the advantage of the use of an optical fiber for remote detection in intracellular measurements is only theoretical, since the low signal level does not permit remote signal collection through the pulled tip. In addition, this approach is not feasible when a large population of cells need to be examined for statistical purposes.
To overcome these problems, we introduce a unique approach for the fabrication of nano-sized optochemical sensors, where the fluorescence sensing dye is entrapped in nano-sized phospholipid vesicles - liposomes (13,14). Nano-sized liposomes containing the oxygen sensitive indicator, tris (1,10 phenanthroline) ruthenium chloride in their internal compartment have been prepared, and tested for their oxygen sensing capabilities in aqueous solution. A standard injection technique, where a lipid mixture, consists of dimyristoylphospatidylchoine, cholesterol, and dihexadecyl phosphate (molar ratio 5:4:1) all dissolved in isopropanol is injected to an aqueous solution of 5 mM Ru(phen)3 under vortexing is used to prepare the liposomes. A high uniformity of the liposomes is realized by extruding them back and forth through a 100 nm sized polycarbonate membrane. TEM and digital fluorescence images of the dye encapsulating liposomes (figures 3a, 3b) show that the liposomes are unilamellar , round in shape, and maintain high structural integrity, with an average diameter of 72 nm. The liposomes show high stability with respect to dye leaking at room temperature, and high photostability when exposed to the excitation light.
The oxygen response of individual dye encapsulating liposomes is studied using digital fluorescence imaging microscopy. Individual fluorescent liposomes are used to monitor the enzymatic oxidation of glucose by glucose oxidase. Depending of the activity of the enzyme solution (known), and the glucose concentration (unknown), a fluorescence increase is observed during the course of the enzymatic reaction, resulting from the decrease in the level of molecular oxygen in the sample. The glucose concentration is determined using kinetic analysis.
The liposome-based oxygen and glucose sensor is the first chemical sensor that can be truly described as a nano-phase material. The absolute limit of detection of the liposome based sensor is at the sub-atomol range, an order of magnitude lower than absolute limit of detection of previously reported submicrometer sized sensors, and 6-7 orders of magnitude lower than the absolute limit of detection of conventional micrometer sized sensors.
The new methods for glucose sensing that are described in this paper offer several advantages compared to conventional glucose measurement systems.
First, miniaturized sensors are less invasive compared to existing sensors for implantation purposes reduced size may lead to increased biocompatibility. Second, miniaturized sensors show a faster response time, in the sub-second range, compared to existing devices. Third, optochemical devices that combine enzymatic selectivity are less sensitive to environmental interferences, compared to electrochemical devices. And finally, the decrease in sensor size down to the nano-sized range, opens the possibility for a true non-invasive intracellular oxygen and glucose analysis.
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