Finding Photonic Ways to Monitor Blood Sugar

Spectroscopic and fluorescence techniques might someday take the sting out of self-checking by diabetics.

People afflicted with diabetes mellitus do not naturally produce enough insulin to process the sugars that they ingest into useful energy for their cells. Instead, they must regulate their sugar intake, increase their level of exercise and — if they are among the 5 to 10 percent of diabetics who have Type 1 of the disease — inject insulin every day. Furthermore, they should regularly monitor their blood sugar — the amount of glucose in their blood serum.

Unfortunately, current methods for home-based glucose monitoring require using sharp lancets to draw blood — typically from a fingertip. Testing blood sugar in this manner is often uncomfortable (even painful) and inconvenient, which discourages diabetics from regular monitoring. To address this disadvantage, investigators are looking into new methods that can continuously measure blood glucose levels with minimum invasiveness.

A group of chemists and biomedical engineers at Northwestern University in Evanston, Ill., is using surface-enhanced Raman spectroscopy (SERS) to develop a method for glucose monitoring. In this technique, proximity of a tested material to a metallic surface (usually silver or gold) enhances the Raman scattering of laser light directed onto the material, thus improving the spectrographic analysis.

However, according to team members Olga Lyandres and Nilam Shah, the researchers had to overcome the fact that glucose does not bind to a bare metal surface. To do so, they used a self-assembled monolayer composed of decanethiol and mercaptohexanol. The monolayers, they said, form well-ordered layers with uniform thickness and surface properties that can be tailored to particular applications.

The investigators chose decanethiol and mercaptohexanol because of their hydrophobic and hydrophilic natures, respectively, which help to pull glucose out of an aqueous solution and localize it within a few nanometers of the enhancing electromagnetic field generated near the metallic surface. The monolayer also aids the sensitivity of the technique by helping exclude nonglucose molecules.

The researchers report in the Oct. 15 issue of Analytical Chemistry that they employed the Raman technique to monitor the blood glucose levels in live rats. They created SERS-active surfaces by depositing a 200-nm-thick silver film over an 18-mm-diameter copper disk that was coated with a solution containing 390-nm-diameter spheres. They then incubated the coated plate in decanethiol and mercaptohexanol, producing a self-assembled monolayer on each surface.

Researchers at Northwestern University have developed a glucose sensor based on surface-enhanced Raman spectroscopy using silver nanoparticles to boost the Raman signal. They tested the technique by surgically embedding a sensor into a rat and measuring the Raman spectra through an implanted optical window. Reproduced with permission of Analytical Chemistry. © 2006 American Chemical Society.

They implanted the plate into a rat by removing the skin and placing the plate beneath a transparent window through which they could measure the Raman spectra. To vary the glucose concentration in the rat, they intermittently injected the animal with a glucose solution over several hours. They acquired SERS spectra using a 785-nm Ti:sapphire laser from Spectra-Physics of Mountain View, Calif., at 50 mW. For comparison with current home-monitoring methods, they also used a consumer glucometer to measure blood glucose levels. According to Lyandres and Shah, they chose the wavelength because it is compatible with in vivo studies, and it reduces the fluorescence of other biological substances that could create an interfering signal.

The graph shows the time course of in vivo glucose measurement. Glucose infusion began at t = 60 min (arrow). Triangles represent measurements made using a consumer-brand blood glucometer; squares, measurements made with the SERS-based sensor. The inset shows a typical in vivo spectrum compared with a typical ex vivo spectrum of the same surface. Reproduced with permission of Analytical Chemistry. © 2006 American Chemical Society.

They found that both the glucometer and the SERS method effectively tracked changes in glucose concentration. Moreover, they noted that changes in concentration as fast as ∋30 s were detectable with the Raman technique because the binding of glucose to the monolayer was rapidly reversible.

Using hydrogels

The investigators are looking to refine the accuracy of their approach as well as to explore other substrates, such as inorganic capture layers and hydrogels, to aid glucose trapping and localization.

Comprising primarily water, hydrogels increasingly are becoming important to biological studies because they are nontoxic, resist degradation and readily immobilize molecules that can act as biosensors. Now researchers at the University of California, Santa Cruz, are using hydrogels as a thin-film substrate to carry a fluorescent dye along with quencher molecules that change the dye’s fluorescence when in the presence of glucose.

The investigators, led by Bakthan Singaram, report in the Oct. 10 issue of Langmuir that they used a hydrogel that incorporated poly(2-hydroxyethyl methacrylate) as their substrate material. Team member Soya Gamsey, who is now at GluMetrics Inc. in Irvine, Calif., said that they used a hydrogel because it constrains the dye and quencher molecules so that they do not leach into the glucose solution, but it does permit the glucose itself to pass through.

Besides the dye, which had an excitation wavelength at 490 nm and an emission at 540 nm, the scientists used viologen, a permanently charged salt of 4,4’-bipyridine that quenches a dye’s fluorescence. They combined the viologen with boronic acid, which acts as a glucose receptor but does not affect the salt’s quenching ability, then put the compound and dye into the hydrogel.

In the presence of viologen, the fluorescence of a dye molecule is quenched (left). When glucose is exposed to the boronic acid attached to the viologen, quenching stops and the fluorescence intensity increases. This principle enabled investigators at the University of California, Santa Cruz, to create a hydrogel-based glucose sensor.

The key to the technique, according to Gamsey, is that glucose infused into the hydrogel binds to the receptor, which decreases the viologen’s quenching ability, thereby restoring the dye’s fluorescence. The investigators then measured the fluorescence and calculated the glucose concentration from that data.

They tested the method using two setups. In one, they excited the infused hydrogel via front-facing illumination created with a 490-nm xenon discharge lamp with 7.3 W of average power at 50 Hz. They gradually added various amounts of glucose to the gel and measured the resulting increase in fluorescence with a spectrometer from PerkinElmer of Wellesley, Mass.

To test the hydrogel technique’s ability to work efficiently in a compact system, the researchers constructed a platform comprising an optical fiber that is encased in a glass tube and capped with the embedded hydrogel. Reproduced with permission of Langmuir. © 2006 American Chemical Society.

They found that the hydrogel displayed a high dynamic response to glucose concentrations within 45 to 360 mg/dl, a range that is especially relevant to diabetics. By decreasing the glucose concentration with the hydrogel and observing the resulting reduction in fluorescence, they also demonstrated the reversibility of the system.

“It is important for [the system] to be reversible,” Gamsey said, “because physiological glucose levels in diabetics and in critically ill hospitalized patients rise and fall. The sensor must be efficient at tracking peaks as well as valleys in glucose concentrations.”

The investigators then devised and tested a miniature hydrogel-based sensing system composed of fiber optics. They threaded one end of an optical fiber through an epoxy-filled glass tube, attached a disk comprising the hydrogel to the end of the tube assembly and immersed the hydrogel-capped end in a cell through which glucose solution flowed.

They connected the other end of the fiber to an Ocean Optics Inc. spectrophotometer equipped with a blue LED light source that provided an excitation wavelength centered at 470 nm. As with the front-facing illumination setup, the scientists found that changes in the fluorescence intensity corresponded directly with glucose concentration. Better still, they saw that the fiber optic system provided faster response times.

In addition, they tested the fiber optic setup’s ability to provide a stable signal throughout extended exposure to glucose, which could indicate the potential for continuous monitoring in diabetics. They exposed the hydrogel sensor to a high concentration of glucose for 36 hours, noting that the fluorescence intensity did not significantly change during this time.

The researchers are hoping to improve the system’s selectivity by finding a way to prevent the boronic acid from binding to molecules that are structurally similar to glucose — such as fructose — which affects the fluorescence signal. They also are modifying the porosity of the hydrogel to attempt to further improve the response time.

“These are the two main hurdles which must be overcome before the system becomes clinically viable,” Gamsey said.