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Raman spectroscopy detects many analytes at once

Jun 2007
Technique could save time and money for clinical laboratory tests

Kevin Robinson

Laboratory testing provides crucial information that physicians use to detect problems, to determine treatment and to monitor treatment effectiveness. Each year, clinical laboratories perform more than 600 million cholesterol tests, 300 million urinalysis tests and a variety of others, such as glucose tests. Although clinical laboratories are mostly automated, in many cases only a few tests can be run on a single sample. An optical method that could test for a large number of analytes at once could improve efficiency and lower costs. Now, researchers at the University of Rochester in New York have overcome key challenges in using Raman spectroscopy for blood testing and urinalysis. Their system can test blood for 11 analytes and can test urine for two, with each test taking only one minute.


Researchers have developed an optical system for analyzing blood serum or urine that involves a liquid-core optical fiber (LCOF), an NIR diode laser, a dichroic mirror (DM), a spectrograph and a CCD to probe the Raman spectrum of the sample. In addition, they use a white light (WL) source and a power meter (PM) to record the absorption spectrum, which then is used to improve the accuracy of the Raman data. (EF = edge filter). Reprinted with permission from Applied Optics.

According to Andrew J. Berger, an associate professor of optics at the university, who, with doctoral student Dahu Qi published a paper on the system in the April 1 issue of Applied Optics, analytes were chosen based on common laboratory tests. “These are major analytes that any physician would screen for in a patient’s blood. We tried to correlate our Raman spectra with every chemical on the list.”

Sample forms core

The system is based on Raman spectra, but with a couple of slight twists. Instead of being housed in a cuvette, the sample forms the core of a liquid-core optical fiber — a thin, hollow tube made of Teflon-AF with a lower refractive index than the sample itself; this creates a waveguide that increases the interaction between the laser light and the sample and that channels the emitted Raman signal to a single exit point. The fiber is 30 cm long, with an inner diameter of 600 μm and an outer diameter of 800 μm.

Signal strength poses one of the biggest challenges for Raman spectroscopy in many applications. Because only about 1 in 1000 photons undergoes Raman scattering, other types of scattering and background noise can drown out the Raman signal. Thus, only with the advent of increased laser power did the method become viable.

Because the enhancement from the liquid-core optical fiber is dependent on light absorption of the sample — with more absorption leading to less signal — it poses a problem with biological fluids, which may vary in the way they absorb light.

“We’d have two different samples with the same amount of protein and not get the same strength of signal,” Berger said. “If we had two samples of blood serum, maybe one sample would be a little pinker due to a few ruptured red blood cells. Then we wouldn’t get the same signal strength.”

To overcome this challenge, the researchers had to calibrate the absorption for each sample. They realized that their experimental setup would allow them to use an off-the-shelf spectrometer to do so by assessing the light absorption of the sample before conducting the Raman analysis.

Once they know the absorptivity of the sample, they plug it into an analytical expression that describes how absorption affects the Raman enhancement caused by the liquid-core optical fiber. By inverting this expression, they arrive at a term that is linearly proportional to the concentration of the chemical but is independent of the sample’s absorptivity.

Analyte testing

In addition to the liquid-core optical fiber, the setup includes a spectrometer from Ocean Optics, a power meter and a white light source coupled onto one end of the liquid-core optical fiber with solid-core optical fiber. On the other end, they coupled a Process Instruments 830-nm diode laser, a Kaiser spectrograph and an Andor CCD array via a dichroic mirror and an edge filter.

The researchers tested blood for 11 analytes: total protein, cholesterol, LDL and HDL levels, glucose, triglycerides, albumin, bilirubin, blood urea nitrogen, globulin and CO2. They tested urine for urea nitrogen and creatinine. In all cases, they compared their results with those from conventional laboratory testing. Ions such as calcium, sodium and potassium have no vibrational spectrum and so cannot be quantified using the system.

The investigators could not detect the presence of creatinine in blood, likely because it is present at very low concentrations. However, they determined that for eight of the 12 analytes, the error is limited by the error rate of the reference analyzer. They also demonstrated that, for eight of the analytes, the extra step of calculating the reference absorption significantly reduced the error rate of the system.

To get the system ready for clinical testing, Berger said that the scientists must improve the way the sample is delivered into the fiber. “Right now, using a bulky syringe and manual injection, we sometimes get air bubbles that disrupt the measurement.” A little “dedicated work,” he said, could resolve that issue. They also have to establish the best way to clean the tubing and to determine how many samples can be measured before it needs to be replaced. “These are probably the biggest practical issues that we haven’t dealt with,” he added.

Berger said that the researchers are looking at applications and continuing to investigate the technique’s potential. “We’re looking for specific applications to serum and urine analysis. One thing we’re considering is in-home monitoring of urine content, to make sure patients are taking certain medications. Some of our urine spectra had very high outlier peaks. We suspect these were from medications, so we need to investigate more.”

Although the Raman technique cannot replace all the tests done by conventional analysis machines, the method could save laboratories money, Berger explained.

raman spectroscopy
That branch of spectroscopy concerned with Raman spectra and used to provide a means of studying pure rotational, pure vibrational and rotation-vibration energy changes in the ground level of molecules. Raman spectroscopy is dependent on the collision of incident light quanta with the molecule, inducing the molecule to undergo the change.
Change of the spatial distribution of a beam of radiation when it interacts with a surface or a heterogeneous medium, in which process there is no change of wavelength of the radiation.
BiophotonicsFiltersmirrorsopticsRaman spectroscopyResearch & Technologyscatteringspectroscopy

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