ISSN(Online): 2319-8753

ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

Design of a Low Cost Single Beam UV/VIS

Spectrophotometer for DNA Measurement
Alakhib A.Abdelbary1, Abdelrahim Elobied Ahmed2
Head, Department of Maintenance and Engineering, Central Laboratory, Khartoum, Sudan1
Associate Professor, Department of Electronic Engineering, Sudan international University, Khartoum, Sudan2

ABSTRACT: The objective of this research is to design and build a complete simple and low cost spectrophotometer
for the DNA measurement. The designed system consists of :two light sources deuterium and halogen lamp ,a
monochromator based on Fastie Ebret design ,a photodiode, a chopper amplifier circuit and a data acquisition system.
The system is controlled using an Atemga microcontroller .the experimental results show good wavelength accuracy.

KEYWORDS: Chopper amplifier, DNA, Photodiode, microcontroller, Spectrophotometer.

I. INTRODUCTION

DNA (deoxyribonucleic acid) is a polymer found in all living cell, it contains all genetic information needed
for controlling cellular growth and development. DNA contains the genetic information necessary for the production of
other cell components [23].
DNA has been the subject of many physical and chemical investigations, leading to discovery of genetics code
and genome sequencing, this effort lead to identification of point mutations and their correlation to pathologies such as
cancer and it is a major target for drug interaction. Thus it is very important to explain the factors that determine
affinity and selectivity in binding molecules to DNA [23]. From this point of view the expansions and technological to
detect DNA are very important
Spectrophotometers can be used to estimate DNA and RNA concentration and to analyze the purity of the
sample. Christian and Wartburg made the first measurement using spectrophotometer for nucleic acid at 260-280nm
[20].
Spectrophotometery is more and often the method of choice in the analysis of biomedical substance, assessment of
food quality, physical properties of various objects, classification and monitoring of optical telecommunication links
[22]
Spectrophotometer is a quantitative method uses the absorbency of light by substance and takes advantage of
two laws of the light absorbency [25]. Lambert law proposed that the proportional of light absorbed by a medium is
independent of the intensity of incident light [26]and Beer stated that the absorption of light is directly proportional to
both the concentration of the absorption medium and thickness of the medium [25] called path length and allow the
measuring sample of different path lengths.
Absorbency=concentration× pathlength (1)
The major drawback of the absorbency detection in microvolumes is that the optical pathlength through the
sample decreases as sample volume decrease and this affects the sensitivity [27]. The spectrophotometer is an
instrument used to measure the absorbance of a sample according to beer Lambert law. It contains the light sources,
spectrometer, detectors and display unit. The light sources which can be used include deuterium, halogen, xenon, and
tungsten lamp,etc..
There are three type of spectrophotometer based on the optical beam: single beam, split beam and double beam
[28].The spectrometer act as a wavelength dispersion element, in the spectrophotometer. The heart of the spectrometer
is the monochromator, whose function is to separate the light according to their wavelengths.
The monochromator contains collimation and focusing mirrors, in addition to gratings and slits. The
monochromator forms an image of the entrance slit in the exit slit plane at the wavelength present in the light source
[2]. There are two configurations for the monochromator; Fastie ebert and Czerny –turner configuration [10].

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3194

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

There are a lot of detectors used in spectrophotometers such as photomultiplier tube, charge - coupled devices
and photodiode. The function of the detector is to convert the light (photons) to current or voltage for further processing
[9].The signal received from the detector is the low current signal which requires amplification and signal conditioning
so as to reject different types of noise. This requires developing and building of a signal conditioning and amplifier
circuits. The paper is organized as follows: in section II the system design is presented .This includes the design of
optical and electronic circuits .Section III presents the results obtained from this design. This includes the validation of
the system and it is application. Finally the conclusion and future scope development were discussed in section IV.

II. SYSTEM DESIGN

The system design of the spectrophotometer contains the design of the spectrometer and the photometer .The
spectrometer includes the light source, sample cell, monochromator and slits. The grating in the monochromator is
controlled using a microcontroller.The photometer includes the photodiode, preamplifier and the data acquisition
system.
Figure1.below presents the elements of the spectrophotometer .The light source is deuterium lamp L2D2 from
Hammatsu company, monochromator which contain spherical and concave mirrors, quartz as a sample cell, the
detector is silicon SI1227 photodiode, amplifier circuit based on chopper stabilized operation amplifier and the
computer used as display .

Figure 1. block a diagram of the system setup of spectrophotometer

A. The spectrometer
The optical system uses an enhanced illumination system to increase optical throughput, sensitivity and to reduce the
bandwidth. We have chosen a commercially available light source (UV or VIS), mirrors and holographic grating, as
can be seen from Figure 2. The light source is focused on the entrance slits of the monochromator, then to the off-axis
ellipsoidal mirror which collects light from the entrance slits and refocuses it to the large spherical concave mirror and
to the grating rotated by a stepper .the off-axis ellipsoidal mirror is used to enhance the throughput of the
monochromator due to under filling of the grating that translate the light away from the required position. It collects
light from the entrance slits and refocused it to the large spherical concave mirror. The large concave mirror is used to
let the grating remains fully illuminated and collects all of light [10]. The spherical concave mirror reflects the light to
the plane holographic diffraction grating, the light is dispersed by grating into various wavelengths and the grating is
rotated by a stepper motor in order to make a scan for all the wavelengths to the exit slit.

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3195

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

Figure 2. Fastie Ebret monochromator

The cell unit is designed to meet the challenge of the low quantity of the sample for the DNA or RNA; therefore
we proposed a new type of the cell it which contains two lenses, one on the side for light coming from monochromator
and the other in the opposite side.
The spectral range of the monochromator is determined by the efficiency of the grating and maximum angle of
rotation of the grating. Fastie ebret configuration suffers from many problems such as spherical aberration, coma,
astigmatism and curved focal field. To solve these problems, mirrors free from aberration are used besides an off-axis
mirror. In order to isolate any other wavelengths, the grating is rotated by a stepper motor, the angular position of the
holographic grating depends on the stepper motor rotation and when the grating rotated different wavelength are
directed through exit slit and to the detector.
Table-1 summarizes the specification of the enhanced optical system which has been based on the Fastie Ebret
configuration using holographic grating, the focal length of concave mirror and the angles calculated and measured
from the designed setup,according to [10] the F/value in and F/value out of the system are obtained.
Table-1 the specification of the enhanced optical system design

Type specifications
Type of optical configuration Fastie ebret monochromator
Grating type Plane holographic grating
Grating size 300× 300 mm
Grating grooves 2400 groove/mm
Focal length of concave mirror 500mm
Angle between incident beam and diffracted beam 12°
F/value in 0.57
F/value out 1.52
Wavelength detector SI 1227
Spectral range 190nm-720nm

B. Grating control
A Stepper motor has been used to rotate the holographic grating in order to scan all the wavelengths coming from the
concave mirror. Figure 3.illustrate The control circuit of monochromator contains, stepper motor, an Atmega 16L 8PU
microcontroller in addition to an ULN2003A driver, which is used to drive the stepper motor, it is operates on 5 volt
DC and it has 6 leads with stepping angles 5.625/64 and 2 phases [2].

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3196

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

U1
9
COM
1 16
C2 ATEMGA 16L 1B 1C
2 15
2B 2C
9 22 3 14
RESET PC0/SCL 3B 3C
23 4 13
PC1/SDA 4B 4C
0.1u 13 24 5 12
XTAL1 PC2/TCK 5B 5C
X1 12
XTAL2 PC3/TMS
25 6
6B 6C
11
CRYSTAL 26 7 10
C1 PC4/TDO 7B 7C
40 27 +88.8
PA0/ADC0 PC5/TDI
39 28 ULN2003A
PA1/ADC1 PC6/TOSC1
38 29
PA2/ADC2 PC7/TOSC2
0.1u 37
36
PA3/ADC3
14 C3
PA4/ADC4 PD0/RXD
35 15
PA5/ADC5 PD1/TXD
34 16
PA6/ADC6 PD2/INT0
33 17 100u
PA7/ADC7 PD3/INT1
18
PD4/OC1B
1 19
PB0/T0/XCK PD5/OC1A
2 20
PB1/T1 PD6/ICP1 +5 Volt
3 21
PB2/AIN0/INT2 PD7/OC2
4
PB3/AIN1/OC0
5
PB4/SS
6
PB5/MOSI
7 32
PB6/MISO AREF
8 30
PB7/SCK AVCC
ATMEGA16

Figure 3. the circuit of the stepper motor and the microcontroller

C. photodiode and preamplifier

We select the silicon photodiode SI 1227 from Hamamatsu photonic company as a detector for the system, table -2
summarizes photodiode general specifications required for the setup which contains effective photosensitive area which
receive light from optical system, the wavelength range of the photodiode cover the range from UV to visible and the
shunt resistance is slope of the current to voltage of the photodiode, the ideal photodiode have an infinite shunt
resistance.
Table-2 SI1227 Photodiode specifications

Parameter value
Effective photosensitivity 5.7mm2
Wavelength(nm) 190-1000nm
Short circuit current 3μA
Shunt resistance 20 G Ω

The function of the photodiode is to convert optical signal from spectrometer to electrical signal to be conditioned by
the amplifier circuit.
In this set up the silicon photodiodes type SI 1227 33BQ has been selected ,the selection is based on the
spectral wavelength from(190nm -1000nm) with peak wavelength 720nm and low dark current 5 pA and short-circuit
current between 2 to 3μA[3].
The expected current from photodiode when subjected to the light source is increased directly by the power of
the light source, this given by equation 2. In this experiment the three light sources have been used, deuterium lamp it
is output power 30 watt, halogen lamp 20 watt and LED lamp 0.25 watt.
IMIN = power × photodide area(mm) (2)
Therefore to detect a small amount of the light, the large area photodiode has been selected [12]. The preamplifier
circuit based on the CMOS ICL7650S Super chopper stabilized amplifiers is best choice of low frequency
measurements[31] such as in our experiment ,it has been selected for many reasons first the diode shunts resistance is a
function of a temperature and this increase the noise in the circuit. Second the input biases current doubles every 10º C
rises in the temperature [30]. This has impact on the circuit and the offset voltage output. Therefore the Super chopper
stabilized amplifiers is the right solution, a chopper stabilized amplifiers is a 14 pins IC device, having two amplifier,
main amplifier and null amplifier in one chip, the input signal from the photodiode connected to the main amplifier
thus, it is bandwidth determine the overall signal. The key specifications of the CMOS ICL7650S Super chopper
stabilized amplifiers shown Table- 3 represent the input offset voltage ,which is indicator of voltage errors in the
output, the low input offset voltage is required in the high performance applications, input bias current caused DC
offset and exceeded photodiode current, the high bandwidth increase the noise in the photodiode circuit and the
chopping frequency used to modulate the frequency in order to minimizes the noise and to prevent the errors due to
aliasing.

Table-3The key specifications of the ICL7650S Super chopper stabilized an amplifier

Parameter Value
Input offset voltage 5 μV
Input bias current 10pA
Gain Bandwidth 2MHz
Chopping frequency 200 Hz

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3197

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

Figure 4.Shows CMOS ICL7650S Super chopper stabilized amplifier. In figure 4 U1 is a main amplifier ,the null
amplifier U2 monitors the input offset voltage of the main amplifier and drive it is output voltage to zero by applying a
correcting voltage at main amplifier null pin [31], the correction voltage held by C1 and C2 holds the correcting voltage
of a nulling amplifier and suppress the undesired offset related ripple without disturbing the desired input signal from
the overall output.
U1
main amplifier

U2
null amplifier

C2 C1
1uF 1uF

Figure 4. CMOS ICL7650S Super chopper stabilized an amplifier

The basic chopper stabilized amplifier circuit is given in Figure 5.The ICL7650S chopper amplifier is connected to two
capacitors .1uF for the nulling connections and the combinations of R1, R3, R4 and R5 serve as an anti aliasing filter to
prevent aliasing due to error from chopping frequency, therefore the input frequency must be much less than one half
the chopping frequency of the ICL7650S amplifier. The output resistor 10k ohm and C2 is used to minimize Vout drop
during auto zero cycle.

U2
7
120.0 VOUT

ICL7650S CHOPPER AMP

TSL251RD
7

3
C2 6
2
10k
0.1u
4
1
8

OP-47G
+14 volt

C4

10000pF

R1 R3 D1

1k 1k
DIODE Volts
C6
R4 R6 D2 +88.8

10000pF 1k 10k
DIODE

-14 volt

Figure 5. Chopper amplifier circuit

D. data acquisition and serial interface to computer

To display the data onto the computer we establish interface between computer and amplifier circuit using AVR
Atemga16L 8PU microcontroller , MAX232 and RS232 cable.Max232 is a 16 pin IC having dual RS232 transmitter
and receivers, it requires external capacitors for it is operation as shown in the figure 6. In an asynchronous RS232
communication, data sent and received as frames. Communication between amplifier circuits and computer start by
sending the start bit which is logic 0, sent for the duration of the bit time, then the 8 data bit is sent, communication
stop by sending the stop bit. Our baud rate is 9600 bits and the frequency of oscillator 8 KHz. The speed of a serial
communication depends on the baud rate and the frequency of oscillator.
The serial data is displayed in the personal computer by using a Matlab R2009 software package to plot the data.
Figure 6. Illustrates the interface circuit to the computer, AVR Atemga16L 8PU microcontroller contains software
program code for receiving data from amplifier circuit and transmits it to the computer by using MAX232. The

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3198

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

MAX232 serve as CMOS level converter for RS232, the function of RS232 is to convert the output voltage from the
microcontroller between 0 to 5 volt to RS232 levels.
ATEMGA MICROCONTROLLER
30 8
AVCC PB7/SCK
32 7
AREF PB6/MISO
6
PB5/MOSI
5
PB4/SS
4
PB3/AIN1/OC0
21 3
PD7/OC2 PB2/AIN0/INT2
20 2
PD6/ICP1 PB1/T1
19 1
PD5/OC1A PB0/T0/XCK
18
PD4/OC1B
17 33
PD3/INT1 PA7/ADC7
16 34
PD2/INT0 PA6/ADC6
15 35
PD1/TXD PA5/ADC5
14 36
PD0/RXD PA4/ADC4
37
PA3/ADC3
29 38
PC7/TOSC2 PA2/ADC2
28 39
PC6/TOSC1 PA1/ADC1 C2
27 40
PC5/TDI PA0/ADC0
26
PC4/TDO
25 12
PC3/TMS XTAL2
24 13 22pF
PC2/TCK XTAL1
23
PC1/SDA
22 9
PC0/SCL RESET C1
ATMEGA16

22pF

1 3 MAX232

C1+ C1-
11 14
T1IN T1OUT
12 13
R1OUT R1IN
10 7
T2IN T2OUT
9 8
R2OUT R2IN C5 C6
2
VS+
6
VS-
10u 10u
C2+ C2- C4 C7
4 5 MAX232
10u 10u

Figure 6. Serial interfaces circuits of a microcontroller and computer

III. RESULT AND DISCUSSION

A. The performance of the system
To test the performance of our spectrophotometer and validate the results from it, the calibration of the
wavelength accuracy test is addressed by using holmium perchlorate or holmium oxide filter [29]. The deuterium lamp
(L2D2) has been used for the measurement of emission line; it showed a peak at 489nm as in the figure 7; this is the
same as the emission of deuterium lamp line of the Jasco530 spectrophotometer instrument. This is a very convent and
accurate way to asses that the instrument optics is aligned and working correctly. Figure 7.presents the emission lines
spectrum of the Deuterium lamp L2D2 in the wavelength range between 460-510nm using scan speed 100nm/min.
Wavelength calibration using Duterium lamp L2D2
0.7

0.65

0.6 489nm

0.55
Intensity (mV)

0.5

0.45

0.4

0.35

460 470 480 490 500 510

Wavelength(nm)

Figure 7. Emission spectrum at 489nm line of the Deuterium lamp

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3199

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

The calibration testing for the spectrophotometer is performed to check the wavelength accuracy; the material used to
calibrate and validate the spectrophotometer is the holmium oxide quartz wavelength standard for Unicam limited
company, Cambridge, UK with reference ref/281/D and ref/281/H. The holmium oxide quartz placed in the position of
the sample cell. Figure 8. Shows measurement of the absorbance spectrum at 241.3, 279.1, 288.8, 334.0 and 359.9nm
of holmium oxide quartz, by using both Deuterium and halogen lamp. This test of the wavelength accuracy
measurement are performed by setting the wavelength scanning speed in the 100nm/min and the measurement ranged
between 200nm to 400nm.
wavelength calibration from 200nm to 400nm using NPL Holium ref W281
0.7

0.6

0.5

0.4
Absorption(mV)

0.3

0.2

0.1

-0.1
200 220 240 260 280 300 320 340 360 380 400
Wavelength(nm)

Figure 8. Wavelength calibration in the measurement range of 200 to 400nm.

The other measurement performed as shown in Figure 9.By setting the wavelength scanning speed in the
100nm/min and the measurement ranged between 500 to 700nm. The absorbance peaks in the (mV) showed at the two
wavelengths 535.8 and 639.2 nm.
Wavelength calibration from 500nm to 600nm
Using Holium wavelength standard
0.4

0.35

0.3

0.25
Absorption(mV)

0.2

0.15

0.1

0.05

-0.05
500 520 540 560 580 600 620 640 660 680 700
Wavelengh(nm)

Figure 9. Wavelength calibration in the measurement range of 500nm to 700nm.

B. Application of the spectrometer for the DNA analysis

Nucleic acid shows a strong absorbance in the region of 240-275 nm, it originates from the π to π* transition to the
pyrimidine and purine, this rising the system of the nucleic bases, e bases can be protonated and therefore the spectra of

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3200

 ISSN(Online): 2319-8753
ISSN (Print) : 2347-6710

International Journal of Innovative Research in Science,

Engineering and Technology
(An ISO 3297: 2007 Certified Organization)

Vol. 5, Issue 3, March 2016

DNA and RNA, DNA absorbs UV light at 260 nm to 280nm due to aromatic base within their structure. The
measurements of DNA made using our spectrophotometer setup as shown in Figure 10. Under these conditions,
scanning speed 100nm/min, sample blanked on water using quartz cell 1 cm and the DNA sample volume
concentration is 100μl. First the blank water has been read, then the DNA sample, the reading of the blank water
subtracted from the DNA sample reading in order to obtain the absorbance result of the DNA as shown in figure 10.
The peak as presents in Figure 10.Iindicates the presented of the DNA in a sample, it contains Purine (Thymin,
cytosinee and uracil) and pyrimidines(adenine and guanine),the absorance peak showed at 275nm.
DNA sample
400

350

300

250
Absorption(mV)

200

150

100

50

-50
200 220 240 260 280 300 320 340
Wavelength(nm)

Figure 10. Absorbance profile of DNA samples from 200 to 340nm

IV. CONCULISION

An enhanced low cost simple spectrophotometer was designed and built. The spectrometer is based on fastie
Ebert configuration and controlled using a microcontroller. The spectrometer optical part was aligned correctly, and
this is validated by measuring emission lines of deuterium lamp. The wavelength accuracy testing shows good accuracy
by using standard holmium quartz.
A successful prototype of the circuits of the grating control, chopper amplifier and the data acquisition system has been
designed from the simple and low cost components.

REFERENCES

[1] Fastie, W.G. and W.M. Sinton," Multiple Diffraction in Grating Spectroscopy", JOSA, 44(2): p. 103-107,1954.
[2] Shukla, R., et al., " Design and Development of 0.5 m Scanning Monochromator and its Application to 15N Analysis". BARC Newsletter,
p.12-19,2005.
[3] DataSheet, H.P., Si-Photodiode S6428-01, S6429-01, S6430-01, S7505-01 Hamamatsu Photonics KK, cat. no. KSPD1013E04, 2003: p. 690-
756.
[4] Witte, F., K.A. Makinwa, and J. Huijsing, ”Dynamic offset compensated amplifiers", Springer Science & Business Media 2009.
[5] Giménez, A. J., R. F. Farías, et al. "Control System Development for a Raman Spectrometer Using Microcontroller Technology", Journal of
applied research and technology ,12(1): 139-144,2014.
[6] Grün, M., R. Müller, et al." Model based control of series elastic actuators. Biomedical Robotics and Biomechatronics (BioRob)" IEEE, 4th
IEEE RAS & EMBS International Conference on, 2012.
[7] Guide, P. "Amplifiers and Comparators."
[8] Joshi, K." Fabrication and Characterization of a Perfusion Bioreactor for Interface Tissue Engineering", 2013.
[9] Khandpur, R. S. (2007). Handbook of analytical instruments, Tata McGraw-Hill Education.
[10] Lerner, J. and A. Thevenon., "The optics of spectroscopy", Horiba-Jobin-Yvon tutorial: http://www. spexjobinyvon.
com/SiteResources/Data/Templates/1divisional. Asp,1988.
[11] Menolfi, C. and Q. Huang "A fully integrated, untrimmed CMOS instrumentation amplifier with submicrovolt offset." IEEE Journal of Solid-
State Circuits,vol. 34(3),pp. 415-420,1999.
[12] Moghimi, R., "Seven Steps To Successful Ultralow Light Signal Conversion"
[13] Oguz, O,"A Lock-In Amplifier for Fluorescent Light Detection", 2002.
[14] Photonics, H,"Photodiode Technical Information" Technical notes, 2012.

Copyright to IJIRSET DOI:10.15680/IJIRSET.2016.0503069 3201