1. INTRODUCTION:

Diabetes is a major disease of concern due to the lack of exercise and poor diet practices. World Health Organization has stated that the count of diabetic people is expected to grow from 246 million in 2006 to 380 million by 2005 and this figure would even rise to 438 million by 2030. So it’s the right time for the Researchers to design a continuous blood glucose monitoring system that makes salutary alertness among the people about the terrifyingly growing disease of concern, so that people are expelled from becoming diabetic.

The present day technologies such as Spectrophotometric measurement or use of lancet devices require fresh blood samples of few millilitres / few drops which limit continuous monitoring. These methods are invasive and seem to be annoying and eluded by people due to the ins and outs such as pain involved; time consumed and increased recurring cost per measurement and also potential risk of spreading diseases like Hepatitis, HIV through interaction of needle with biotic fluids of human skin. All these aspects makes prime craving for what is called Non-invasiveness. Diabetes mellitus is a condition of deficiency rather than a disease because people are said to be diabetic when their Pancreas no longer produces insulin or when cells in the body stop responding to insulin. Glucose Diagnosing can be carried out under three ways: - Invasive technique, minimally invasive technique and Non-invasive technique. NI blood glucose measurement is mostly expected to take over the era, because it involves no pain. This can be accomplished by two ways either optically or Non-optically. Non-optical means of measuring blood glucose involves acquiring glucose values from Sweat, breathe, tears or urine which does not directly reflect blood glucose value and hence most of the research works concentrates on optical means.One of the challenging tasks of Non-invasiveness is detection of weak blood signals that lose energy through intervening tissues and in addition separating glucose’s information from other overlapping constituents with much higher concentration such as protein, urea, uric acid, haemoglobin, water, etc… This task can be performed by using high quality transducers / sensors, so that the sensors can measure either direct chemical structure of glucose or indirectly by measuring blood sugar based on secondary terms like temperature or PH changes ^{[1, 2]}. The great volume of research works at present concentrates on developing a NI device using one of the following techniques such as: - Reverse Iontophorosis, Polarimetry, Metabolic Heat Conformation, Ultrasound Spectroscopy, Thermal Emission, Electromagnetic technique, Photo acoustics, Raman Spectroscopy and Bio-impedance Spectroscopy. Few of the works concentrates on Multivariate analysis for monitoring of blood glucose. Out of the above mentioned techniques Complex Impedance and Optical Spectroscopy shows most promising results ^[3]. Apart from the technology one must also rely on several influencing factors like skin temperature, pressure, sampling duration, breathing artifacts, blood flow rate, body movements, tissue thickness, surface roughness, sweat and skin colour while going for calibration of such device ^[4]. In this paper two of the optical techniques namely Absorption Spectroscopy and Photo acoustics Spectroscopy because of their superiority, have been compared and considered for refinement in order to improve the efficiency of the equipment. This paper deliberates the design and outcomes of using an Ultra Violet – Visible Light (UV-VIS) Spectrophotometer in section 2 [for Studying the absorption property of glucose thereby selecting the correct Light Source of proper wavelength, which is to be nominally employed in the design part] and the upshots of Absorption Spectroscopy (AS) Vs Photo Acoustic spectroscopy (PAS) technique is roofed in section 3 and discuses the results obtained in the concluding fraction.

2. UV-VIS SPECTROPHOTOMETER:

UV-VIS Spectrophotometer refers to the absorption or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent (near-UV and Near-Infrared [NIR]) ranges. The Beer-Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.^[10]

When a beam of monochromatic light falls upon a substance, the fraction of the light absorbed is a function of the concentration of the substance and the path length. The UV-VIS spectrophotometer measures the intensity of light passing through a sample ({\displaystyle I}I), and compares it to the intensity of light before it passes through the sample ({\displaystyle I_{o}}I₀). The ratio {\displaystyle I/I_{o}}(I/I₀) is called the transmittance (T), and is usually expressed as a percentage (%T).

Thus,

T = I/Io ------------ (i)

Mathematically, it is found that

ln Io - ln I = Kb ------------- (ii)

Where,

K = a proportionality constant, characteristic of the nature of the absorbing solute.

b = path length.

Intensity of the transmitted light decreases logarithmically as the path length increases arithmetically. of greater interest is the dependence of the transmitted light on the concentration of absorbing solute, Beer found that increasing the concentration of absorber had the same effect as increasing the path length 'b'. Thus the proportionality constant 'k' is in turn proportional to the concentration 'c' of absorbing solute, or

k = ac ---------- (iii)

Thus equation (ii) is rewritten as

log (Io/I) = abc ---------- (iv)

Where, 'a' is another constant, which also takes care of expressing the equation with base of ten logarithms. Equation (iv) is known as Lambert-Beers Law. Substituting equation (i) in equation (iv) we get

log 1/T = abc

This gives a relationship between Transmittance, T and concentration 'c'. However, the relationship between the concentration 'c' and the log of inverse of ‘T’ is inconvenient in quantitative analysis. Therefore, another quantity is introduced, called Absorbance, A as

A = log 1/T ---------- (v)

Thus

A = abc --------- (vi)

Or in other form the absorbance, {\displaystyle A}(A), is based on the transmittance and given as:

A = - log (%T/100%). {\displaystyle A=-\log(\%T/100\%)}

The above law is of fundamental importance to Spectrophotometric analysis. The basic parts of a spectrophotometer are a light source, a holder for the sample, a diffraction grating in a monochromator or a prism to separate the different wavelengths of light, and a detector. UV light is sourced by Deuterium arc lamp and VIS light is sourced by Tungsten-Halogen lamp, automatically switched at user-selected wavelength The radiation source is often a Tungsten filament of range: 300-2500 nm, out of which the model no: 119 used by us covered wavelength range from 200nm to 1000nm with a setting accuracy of ± 0.5 nm, The detector is typically a photomultiplier tube, a photodiode, a photodiode array or a charge-coupled device (CCD). In this case a high sensitivity and wide range solid state silicon photodiode is used as the detector. The measurements are presented in %T (percent Transmission), ABS (ABSORBANCE) or CONC (Concentration) terms. A 5-position automatic sample holder is provided for quick comparison of samples. Finally, the transmitted light from the cells reaches the Photodiode. The electrical output of the photodiode is linearly proportional to the intensity of light falling on it and represents the measurement being taken. The grating spectral order interference is eliminated by order cut-off filters. A sine bar linkage rotates the grating to get accurate wavelength, which in turn is moved by a stepper-motor driven screw-rod mechanism. The movement of the stepper-motor is monitored/controlled by the computer-controlled electronic circuits. The UV-visible spectrophotometer can also be configured to measure reflectance^{[5, 6].} In this case, the spectrophotometer measures the intensity of light reflected from a sample ({\displaystyle I}I), and compares it to the intensity of light reflected from a reference material ({\displaystyle I_{o}}I₀). The ratio {\displaystyle I/I_{o}}(I/I₀) is called the reflectance, and is usually expressed as a percentage (%R).

Figure1. Transmittance Vs. Reflectance

The scanning monochromator moves the diffraction grating to "step-through" each wavelength so that its intensity may be measured as a function of wavelength. Fixed monochromators are used with CCDs and photodiode arrays. As both of these devices consist of many detectors grouped into one or two dimensional arrays, they are able to collect light of different wavelengths on different pixels or groups of pixels simultaneously. A spectrophotometer can be either single beam or double beam. In a single beam instrument all of the light passes through the sample cell. {\displaystyle I_{o}}I₀ must be measured by removing the sample. This was the earliest design and is still in common use in both teaching and industrial labs. In a double-beam instrument, the light is split into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample.

Figure2. Simplified Schematic of a double beam UV – VIS Spectrophotometer

Samples for UV-VIS spectrophotometer are most often liquids, although the absorbance of gases and even of solids can also be measured. Samples are typically placed in a transparent cell, known as a cuvette. Cuvettes are typically rectangular in shape, commonly with an internal width of 1 cm. (This width becomes the path length, {\displaystyle L}L, in the Beer-Lambert law.) Test tubes can also be used as cuvettes in some instruments. The type of sample container used must allow radiation to pass over the spectral region of interest ^[9]. The most widely applicable cuvettes are made of high quality fused silica or quartz glass because these are transparent throughout the UV, Visible and NIR regions.

HEALTH EFFECTS OF UV RADIATION:

Small amounts of UV are beneficial for people and essential in the production of vitamin D. UV radiation is also used to treat several diseases, including rickets, psoriasis, eczema and jaundice.

Figure3. Relationship of exposure to UVR and Burden of Disease

Prolonged human exposure to solar UV radiation may result in acute and chronic health effects on the skin, eye and immune system. But this methodology is used only to select best light source wavelength for better absorption by glucose on the test sample and not subjected to be exposed on direct human skin, hence continuous exposure is avoided and results in no harm ^[7].

GLUCOSE ABSORPTION PROPERTIES:

Absorption properties of glucose have been studied by means of UV-VIS spectrophotometer. Readings obtained from the spectrophotometer implies that glucose has good absorption properties. The readings will show the absorption rates for different glucose concentrations. The test has been undertaken for low, medium and high concentrations. From the above readings, it is clear that when the concentration increases the absorption rate also gets increased.

GLUCOSE ABSORPTION RATE FOR DIFFERENT WAVELENGTHS:

Glucose has different absorption rates for different wavelengths. Glucose absorption rates have been studied for various wavelengths in between 350-950nm. This is to find out at which wavelength, the glucose has good absorption properties. The graph below shows the glucose absorption properties for wavelengths between 350-750nm.

Absorption rate decreases when the wavelength increases, and again it increases at 950nm wavelength. From the above studies it is clear that, glucose has shown good absorption characteristics for to the wavelengths 360nm and 950nm. For the biomedical project, higher wavelength laser cannot be used. Therefore, 360nm wavelength laser was chosen for the design part. The images of various Light sources that can be applied at the transmitter part of our circuit are:

Figure 9. Fiber Output Laser Diode Bar Modules

This is UV-Visible fiber light source for complete range of portable systems. There are two types, L10671 and L10290. The L10671 contains S2D2 (Stable and Small Deuterium) lamp and tungsten lamp which emits range of 200nm to 1600nm. The L10290 contains high brightness deuterium lamp and tungsten halogen lamp which emits range of 200nm to 1600nm.

3. UPSHOTS of AS Vs PAS:

In PAS technique, the glucose in blood is agitated by short spell LASER pulse. Light absorption produces stress in the internal tissues, ensuing in heating of the medium as a result of which a pressure wave is generated that travels outwards that can be picked up by a transducer. The amplitude of the pressure wave gives the concentration of glucose ^[8]. Piezoelectric Transducer made of Lead Zirconate Titanate (PZT) has been made use of for detecting the PA signal . PA signal can also be given the provision of being stored and processed using a Digita Storage Oscilloscope (DSO) at specific intervals. Output of the transducer is amplified by Low Noise Amplifier (LNA). Baseline pre-processing is done to filter random noise and interferences by averaging the PA signals over 1024 frames to achieve appreciable Signal to Noise Ratio (SNR). Oral Glucose Tolerance Test (OGTT) was carried out on several entities for testing the system functionality and the PA signals were recorded at regular intervals and processed using Matlab to obtain the peak value. Absorption Spectroscopy is a workhorse expertise extensively used in many research fields mainly due to its innovative approach. This technique involves light source to impinge light on to the subject under study and a photo detector for processing the sensed signal. This method of measurement seems to be attractive and smart because of the advantages like: easy and convenient operation, low cost, safety, increasing availability of LASER light source and real time assessment. The absorption property of glucose was studied using UV-VIS Spectrophotometer and the selected wavelength LASER source from that technique was employed and a photo diode was used as detector and readings were obtained. Two different sensor readings [ PAS using Piezoelctric YTransducer and AS- using normal LASER source] were compared and concluded that due to over pre processing and baseline post processing procedures piezoelctric component was eliminated and the entire design was made considering two absorption LASER sources and Photo diodes. Values of two photo diodes were averaged and given to the Embedded Controller to get the digital value. The digital equivalent was converted into direct blood sugar value in mg/dL by normal extrapolation and interpolation methods and was used to frame a Look-Up Table (LUT) which provided a correlation coefficient, R of 0.71, and hence the direct Sugar value in mg/dl was displayed.

4. RESULTS AND DISCUSSIONS:

When a person tries to diagnose his/her blood sugar level, he/she must initially switch on the LASER light sources circuitry operating around 350nm wavelength and place the finger on the sensors platform and the reflected light intensity will be detected by the photodiodes. The photodiodes convert the received light intensity into electrical analog voltage. In order to get larger variants and to set apart, it was necessary to produce the digital equivalent of the analog voltage which was accomplished using an ADC. The setup was validated for about 35 patients in JEBI Diagnostic Centre at repeated intervals for different categories of people – Normal, Hyperglycaemia and hypoglycaemia persons and it was compared with Spectrophotometric value obtained from the same lab. To our surprise the peak values likely matched the real values for most of the cases.

CONCLUSION:

The below tabulation will show the results obtained from the hardware setup for about 7 samples out of 35 samples.

Table1. Results Obtained

S.NO	Blood Glucose Level in (mg/dl)	Averaged outputs from Sensors in volts	ADC Value
1	74	4.89	1013
2	89	4.75	1007
3	97	4.77	1002
4	114	4.57	985
5	124	4.56	976
6	97	4.77	1003
7	100	4.70	997

In conclusion this paper reached at the objective of finding the best absorption wavelength of Light source by Blood Glucose using UV-VIS Spectrophotometer and used the same for designing a device that Non-invasively diagnosis diabetes.

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Received on 30.09.2016 Modified on 17.11.2016

Research J. Pharm. and Tech. 2017; 10(1): 91-97.

DOI: 10.5958/0974-360X.2017.00022.1