CHAPTER 3 MEASUREMENT OF GLUCOSE IN BLOOD SERUM
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1 55 CHAPTER 3 MEASUREMENT OF GLUCOSE IN BLOOD SERUM 3.1 INTRODUCTION Glucose is the most important carbohydrate fuel in the body. In the fed state, the majority of circulating glucose comes from the diet and in the fasting state, gluconeogenesis and glycogenolysis maintain glucose concentrations. The blood glucose levels are perfectly maintained under the influence of hormones like insulin and glucagon. However, the hormonal imbalance sometimes may result in abnormalities of glucose metabolism and result in diseased condition (Srikanth et al 2004). Conditions of hyperglycemia (Diabetes) and hypoglycemia result from an imbalance between systemic glucose delivery (endogenous glucose production with or without absorption of exogenous glucose) and glucose utilization (Seshiah and Balaji 1998). Thus, it is important to measure the glucose level in blood of the human in order to identify the normal or abnormal condition. There are several techniques adopted for glucose analysis in clinical practice. The three major basic approaches to the laboratory measurement of blood glucose concentration are (1) reducing method (2) condensation method and (3) enzymatic method (Kenneth Walker et al 1990). In the enzymatic reaction, the enzyme preferentially reacts with the blood glucose forming hydrogen peroxide, which on further reaction results in a colored complex. In the standard colorimetric method, the intensity of the colored complex is directly proportional to the glucose concentration present in the sample. At present there are several optical diagnostic techniques, such as near-infrared
2 56 spectroscopy, optical coherence tomography (OCT) (Larin et al 2007), polarimetry, Raman and fluorescence spectroscopies. These techniques have been tried and tested for monitoring free glucose and glycated hemoglobin levels in blood by Zhernovaya et al (2008). Studies on optical properties of biological samples are now having renewed interest due to their varied technological applications. In the past years the possibility of using biological materials in photonic devices has been reported by many authors (Koyama et al 1994, Birge 1994). This comes as a consequence of focused attention to the optical properties of biomolecules, as part of better understanding of the fundamental events associated to biological processes (Helene and Charlier 1982). Some examples are, the characterization of lipids in body fluid (G omez et al 2004, 2006), study of the nonlinear refraction of vitreous humor in human and rabbit (Rockwell et al 1993), determination of nonlinear refractive index of retinal derivatives (Arandi et al 1997) etc. Interestingly, a method to determine the glucose concentration based on refractive index value has been reported by Yoder-Short (1992) and Zirk et al (2004), (2007) where the measured linear refractive index value is found to vary linearly with respect to the concentration of glucose. Recently, refractive index of water solution of hemoglobin and albumin with different glucose concentration was measured using Abbe refractometer (Zhernovaya et al 2008). Optical nonlinearity has been reported for LDL-Cholesterol (Kroll and Chesler 1994, 1998). As a natural extension, it generated interest to measure the nonlinear refractive index value of the given sample under the exposure of laser beam. Among various techniques developed to measure the nonlinear optical refractive index, the single beam Z-scan technique, proposed by Sheik Bahae et al (1990), is a simple and effective tool. It is a method, which can rapidly measure both nonlinear refraction and the nonlinear absorption in solid and
3 57 liquid samples (Qusay and Palanisamy 2005, 2006, Madhana Sundari and Palanisamy 2006, Ahmad et al 2007). Nonlinear refractive index is proportional to the real part of the third-order susceptibility Re[χ (3) ]. The Z-scan method exploits the selffocusing and defocusing phenomena in nonlinear optical materials. In this method, the nonlinear sample is exposed through the focal plane of a tightly focused Gaussian laser beam and the change in the far field intensity pattern is monitored. For a purely refractive nonlinearity, the light field induces an intensity dependent nonlinear phase and as consequence of the transverse Gaussian intensity profile, the sample presents a lens-like behavior. The induced self-phase modulation has the tendency of defocusing or recollimating the incident beam, depending on its Z position with respect to the focal plane. By monitoring the transmittance change through a small circular aperture placed at the far field position, one is able to determine the nonlinear refractive index. 3.2 METHODOLGY Serum Separation About 1 ml of whole blood sample was drawn and injected into a sterile screw capped centrifuge tube, which yields approximately 0.4 ml of serum (after clotting and centrifuging). Then the centrifuge tube was placed in the upright position in the rack, and the blood was allowed to clot at room temperature for 30 minutes. After the clotting of blood has occurred the tube (with stopper end up) is inserted into the centrifuge. The centrifuge is operated for 10 minutes at the rate of 3000 rpm. Then the stopper is removed and serum from cells was carefully aspirated, using a separate disposable
4 58 pipette for each tube. The collected serum is transferred into the cleaned dry test tube. This serum is further used for measurement of bioanalytes Glucose Sample Preparation About 10 l of serum is pipetted out in a clean and dry test tube. Then, 1ml of reagent {a kit supplied by Merck (Merckotest Glucose based on GOD/POD)} is added to the serum. The solution is mixed well and incubated at 37 o C for 15 minutes. The solution turned to pinkish red color. The principle involved in this reaction is represented as Glucose + O 2 + H 2 O GOD Gluconic Acid + H 2 O 2 2 H 2 O Aminoantipyrine + Phenol POD Quinoneimine + 4 H 2 O The glucose is oxidized by glucose oxidase (GOD) to form gluconic acid with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase (POD) to yield Quinoneimine dye compound Measurements The prepared sample is subjected to optical nonlinearity measurement by Z-scan technique. The Z-scan experiments are performed using a 532 nm Nd: YAG (SHG) CW laser beam (COHERENT Compass 215M-50 diode-pumped lasers) focused by a lens of 3.5 cm focal length. A typical closed-aperture Z-scan curve for the standard glucose solution at incident intensity I ο = kw/cm 2. This normalized transmittance curve is characterized by a pre-focal peak followed by a post-focal valley. This implies that the nonlinear refractive index of standard glucose and serum sample is negative (n 2 < 0). The defocusing effect shown in Z- scan curve
5 59 can be attributed to a thermal nonlinearity resulting from absorption of radiation at 532 nm. Localized absorption of a tightly focused beam propagating through an absorbing sample medium produces a spatial distribution of temperature in the sample solution and consequently results in a spatial variation of the refractive index that acts as a thermal lens resulting in phase distortion of the propagating beam. The nonlinear refractive index (n 2 ) is calculated using the standard relations by Sheik Bahae et al (1990) and Sutherland (1996). = (1-S) 0.25 o (3.1) Tp v where can be defined as the difference between the normalized peak Tp v and valley transmittances ( T p T V ), o is the on-axis phase shift at the focus. S, the linear transmittance of the aperture is given by S= 1- exp(- 2r 2 a / w 2 a ) (3.2) where r a is the radius of the aperture and w a is the radius of the laser spot before the aperture. n o 2 (3.3) kioleff 2 where n 2 is the nonlinear refractive index, k is the wave number ( k ) and L eff = 1 e L.
6 60 2P I o = 2 w o is defined as the peak intensity within the sample at the focus. L is the thickness of the sample, α is the linear absorption coefficient. Further, experiments were also performed with conventional colorimetric method following the standard procedure of Trinder (1969) for the chosen sample. This involves measurement of change in optical density with respect to concentration. These measured results are compared with the results calculated with the present Z-scan technique. 3.3 RESULTS AND DISCUSSION Measurement of Absorbance Spectra The UV-Vis (Ultraviolet-Visible) absorption spectra of the sample are recorded using an UV-Vis spectrophotometer (Perkin Elmer- Lambda35) and the absorption peak is found to occur at 506 nm, as depicted in Figure Absorbance (arb.units) Wavelength (nm) Figure 3.1 UV-Vis spectrum of the standard glucose with reagent
7 Measurement of Nonlinear Refractive Index The result of typical Z-scan normalized transmittance measurement is shown in Figure 3.2. As the concentration of the standard glucose increases, the intensity of the normalized transmittance peak increases whereas the intensity of the valley decreases. In Figure 3.3 (a) the graph shows that the ΔT P-V linearly increases with concentration of standard glucose solution. Similarly in Figures 3.4 and 3.5 nonlinear refractive index and optical density linearly increase with concentration of standard glucose solution. The experiments have been repeated five times and the mean value of the nonlinear refractive index (n 2 ) is calculated from the normalized transmittance values. This calculated value is assumed to be the standard for measurement of unknown glucose content present in blood sample. This can be arrived by plotting a linear graph of concentration of glucose Vs nonlinear refractive index. The nonlinear refractive index value is first measured against the reagent blank solution. The calibration is made with the conventional colorimetric method and the results are tabulated in Table 3.1. Normalized Transmittance mg/dl 90 mg/dl 100 mg/dl Z (mm) Figure 3.2 Z-scan data of the standard glucose
8 T p-v R 2 = Y= x Concentration of Glucose (mg/dl) Figure 3.3 Linear variation of ΔT p-v with glucose concentration n 2 X 10-8 (cm 2 /W) R 2 =0.99 Y= x Concentration of Glucose (mg/dl) Figure 3.4 Linear variation of n 2 with glucose concentration
9 Optical Density (a.u) R 2 =0.99 Y= x Concentration of Glucose (mg/dl) Figure 3.5 Linear variation of optical density with glucose concentration Table 3.1 Nonlinear refractive index (n 2 ) values for standard glucose Standard glucose Concentration (mg/dl) Nonlinear refractive index n cm 2 /W Trinder (1969) proposed that the allowed glucose level in blood sample is mg/dl, below which leads to hypoglycemia and above which leads to hyperglycemia. Hence from the results we incur that the value of n 2 < cm 2 /W leads to hypoglycemia and n 2 > cm 2 /W leads to hyperglycemia and the values in the range between 1.82 and cm 2 /W are considered as normal glucose level.
10 Evaluation with conventional method Many trials have been performed to measure the glucose level with our proposed method. Three samples have been taken randomly. Measurements are taken during fasting and also after food intake (postprandial) for two persons. We could see that the results arrived are in good agreement with those of the conventional colorimetric method as shown in Table 3.2. Hence we could clearly ascertain that the proposed method is on par with the conventional colorimetric method. Generally speaking, this technique can be used in all places where the single beam Z-scan set up is employed. Table 3.2 Serum glucose measurement using colorimetric method and Z-scan method-a Comparison Sample collection Glucose level Glucose concentration(mg/dl) Colorimetric method Z-scan method Random Normal Random Hyperglycemia Random Hypoglycemia Fasting Normal Postprandial Normal Fasting Normal Postprandial Hyperglycemia Each value is the mean of 5 individual observations. The P value (t-test value) is less than 0.01 at 1% significance level.
11 CONCLUSION We have measured the nonlinear refractive index (n 2 ) for the standard glucose and blood samples by employing the single beam Z-scan technique with 532 nm Nd:YAG CW laser. The Z-scan measurements indicate that both standard glucose and blood sample exhibit nonlinear optical properties. The experimental results show that the optical density and nonlinear refractive index (n 2 ) of the given samples being measured with colorimetric method and Z-scan technique respectively are found in good agreement. Thus the nonlinear refractive index value of the given blood sample can be treated as a good quantifying indicator of the glucose concentration.
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