Portable and modularized fluorometer based on optical fiber

Weiwei Yue*,Lei Zhang,Zhenya Guo,Shouzhen Jiang, Chengjie Bai

College of Physics and Electronics, Shandong Normal University, P.R. China, 250014
*Corresponding Author: TEL: +086053186182521.FAX: +086053186182521
E-mail: yuewei@sdnu.edu.cn

ABSTRACT

A portable and modularized fluorometer based on optical fiber was proposed in this work. The fluorometer included a
light emitter diode (LED) light source module (LSM), a sample cell module (SCM), an optical-electrical converter
module (OCM) and a signal process module (SAM). The LEDs in LSM were driven by a constant current source to
provide stable exciting light with different wavelength. The OCM included a modularized optical filter and used a
photomultiplier tube (PMT) to detect fluorescence signal. The SCM was used to locate sample cuvette and could be
connected by optical fibers with the LSM and OCM. Via modularized design, the LSM and OCM could both selected
and replaced based on different fluorescence dyes. In order to improve the detecting dynamic range of the fluorometer,
the SAM could control the light intensity of LED source in LSM, to control the gain of PMT in OCM, and particularly,
four channel signal acquisition circuits with different gain were constructed to collect fluorescence signal
simultaneously. Fluorescein isothiocyanate (FITC) was selected as sample to test the fluorometer. The fluorometer has
shown a high sensitivity with FITC concentration of 10ng/mL and presented a good linearity from 10 ng/mL to 10
μg/mL.
Keywords: Fluorometer, Optical Fiber, LED, PMT

1. INTRODUCTION
Fluorescence photometer was the most routine instrument and has widely application in biochemical, environmental and
medical fields [1-3]. However, conventional fluorescence photometer adopts complex light source and optical system to
get homogeneous exciting light and emitting fluorescence signal, the instrument was generally large in size and was
strict with stability which has resulted in an expensive cost. Therefore, it is valuable to develop a kind of low-cost and
stable fluorometer[4].
Since high energy efficiency in producing monochromatic light and the flexibility for changing wavelength as well as
tiny size, light-emitting-diodes (LEDs) have been widely studied and used as light sources in fluorescence system[5-6].
Yongjiang Dong et al. have developed a fluorescence system by using several light emitting diodes (LEDs) with
different wavelengths as excitation light sources for tea classification and quality assessment [7]. Combined with micro-
electro-mechanical systems technique and microfluidic cell chip analytical methodology,LI Dong-Shun et al. have
constructed a LED induced fluorescence detector for monitoring cells in the suspensions with a low concentration below
40 cell/mL[8].
In order to improve the stability of the fluorescence system, optical fiber has been widely adopted in fluorescence
detection [9-11]. SHI Ying et al. have established an approach to continuously record fluorescent signals of rat cerebral
cortical neurons in vivo, using the novel system composed of fiber-optic probe and fluorescence microscopy[12]. The use
of optical fibers combined with immobilized fluorescence sensors is a challenging analytical chemistry area. Helena et
al. have developed a fiber-optic sensor based fluorescent carbon nanoparticles for Hg(II) ions detection[13].
In this work, a portable and modularized fluorometer based on optical fiber and LED has been presented. The
fluorometer included a LED light source module (LSM), a sample cell module (SCM), an optical-electrical converter
module (OCM) and a signal acquisition module (SAM). In order to improve the stability of the system, optical fiber was
used as light guide media. All the modules were connected by optical fibers. Via this design method, a fluorometer with
characteristics of miniaturization and modularization has been developed in this work.

Ninth International Symposium on Precision Engineering Measurement and Instrumentation

edited by Jiubin Tan, Xianfang Wen Proc. of SPIE Vol. 9446, 94464I · © 2015 SPIE
CCC code: 0277-786X/15/$18 doi: 10.1117/12.2182022

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 2. SYSTEM DESIGN
The fluorometer included LSM, SCM, OCM and SAM as shown in Figure 1. The LSM could generate exciting light
with different wavelength. The exciting light was guided to the SCM by an optical fiber with SMA905 connector. The
fluorescent material was excited and the emitted fluorescence was collected by a fiber collimator in the orthorhombic
direction and guided to the OCM through another optical fiber. The emitted fluorescence was filtered and converted to
electrical signal by a photomultiplier tube (PMT) in the OCM. Through signal amplification and A/D (Analog/Digital)
conversion by the SAM, the relative intensity of the emitted fluorescence was uploaded to an upper computer and
displayed in real-time.

4
1
Signal
Acquisition

Upper Computer

Light Source Sample Cell Optical Converter

Module Module Module
Figure 1. Structure of the fluorescence spectrophotometer
2.1 LED light source module
The LED light source module has integrated several LEDs with different central wavelength as exciting light source for
samples. The LEDs were driven by an adjustable constant current IC module MAX1916. Each MAX1916 could drive
three LEDs with maximum current 60 mA respectively. The drive current of LEDs could be adjusted by a D/A
(Digital/Analog) IC DAC8534 which could generate 4 channels analog voltage controlled by a microcontroller unit
(MCU) STC15f2k60s2. The stability of the LED light source has been tested in the results section in this paper.

U2
SYNC6 SYNC D
5 D
MCU SCLK7 SCLK C
4 C
DIN 3 B
DIN 8
NC 2 A
GND VCC
A
VCC
DAC 8534Modo le

VCC

MAX 1916

Figure 2. LED drive circuit and photograph of the light source module
2.2 Sample cell module
The schematic and photograph of the sample cell module (SCM) have been shown in Figure 3. Incident optical fiber and
emergent light optical fiber were connected with the SCM through standard SMA905 connectors in orthogonal
directions. Micro cuvette was used as sample cell with capacity about 100 μL. All the SCM was oxygenized with black
color in order to absorb the stay light. The background signal and noise were detected to estimate the sensitivity for the
fluorescence signal.

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 SMA905

Figure 3. Schematic and photograph of the sample cell module

2.3 Optical-electrical converter module
The emitted fluorescence was guided through an optical fiber into the OCM. Through a fiber collimator, the fluorescence
was converged to an optical filter. A bandpass optical filter (Semrock,USA) was embedded in the OCM to filter the
exciting light and stray light. According to center wavelength of different fluorescent dyes, the bandpass optical filter
could be replaced.
The filtered fluorescence signal was converted to electrical voltage by a PMT (CH253, HAMAMATSU, JAPAN). The
CH253 PMT module has spectral response range from 180 nm to 900 nm with peak wavelength 400 nm. The gain of
CH253 could be adjusted by a control voltage from 1V to 4.5V. The control voltage could also obtain from D/A module
DAC8534 channel A as shown in Figure 2 and Figure 4. The output voltage signal was amplified and filtered by a four
channel signal acquisition circuit.
Optical PMT
filter
il
SMA905 / \
Output
signal

I \/

(a) Schematic of the OCM (b) Photograph of the OCM

Figure 4. Schematic and photograph of the optical electrical conversion module
2.4 Signal acquisition circuit
Since the fluorescence signal is very weak, the PMT with high sensitivity was selected as photoelectric converter.
However, since the high sensitivity of the PMT, the response range would be decreased. In order to solve the
contradictory of high sensitivity and wide response range, the signal acquisition system included four channels of
acquisition circuit with voltage gain from 60 dB to 120dB. The optical-electrical signal of each channel was collected by
a multi-signal acquisition system simultaneously. Only one channel was selected as final signal based on the actual PMT
signal intensity. The schematic of signal acquisition system was shown in Figure 5.

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 rAmplification \ /
-0 AD
60dB
rAmplification i-, r i
-.
r PMT 1 rLowpass1 -0 80dB
AD
MPU
I -- STM32F
/
Signal Filter
-0
Ámplification\
.r 100dB ,
- AD 103ZET6

Amplification
-0 120dB
-* AD

Figure 5. Schematic of signal acquisition system

2.5 Control software design
The control software included two portions, one was the lower microprocessor software (LMS) and the other was the
upper computer software (UCS). The LMS actuated the operations including controlling LED intensity, adjusting PMT
gain and selecting signal channels. The UCS was human-computer interface which was used to generate controlling
instructions and display the detecting fluorescence signal real time. The control schematic was shown as Figure 6.

PMT -, Multi -signal

acquisition
MPU
STM32ZET6

PMT Gain N

LED
JJJ
DAC8534
DA module
H MCU
STC15f2k60s2
1
Figure 6 Control schematic of the fluorometer

3. RESULTS AND DISCUSSION

3.1 LED intensity and stability
The spectroscopy of the LED was measured by a fiber optic spectrometer (USB2000, Ocean Optics) .The relationship
between light intensity and control voltage was shown in Figure 7. It was shown obviously that the intensity of the LED
could be controlled by the control voltage with good linearity.
5000

4500 - -6-1 V I uowwq

-0- 1.5 V -wnFwp LED wry
!N
C
4000 - t2V
3500 -
-0- 2.5 V
-A-3V
3000 -

tx 2500 -
W 2000 - 5 t0 50

J Caw Vd1119$ IVI

0
460 480 500 520 540 560

Wavelength (nm)

Figure 7. Relationship between light intensity and control voltage

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 3.2 PMT gain
The gain of the PMT could be adjusted by an external controlling voltage from 1V to 4.5V. In order to test the
relationship between PMT gain and controlling voltage, the output signal of PMT and controlling voltage has been
detected as shown in Figure 8. Fluorescein isothiocyanate(FITC) with concentration of 0.1 μg/mL, 1 μg/mL, 5 μg/mL
and 10 μg/mL has been prepared as test samples. For each samples, the curve represented the detected fluorescence
intensity with increase of controlling voltage in Figure 8. It can be seen from Figure 3 that the PMT gain could be
adjusted by the controlling voltage exponentially.
40000

t 0.1 ug/mL
Ñ 35000 -
c -r 1 ug/mL
5 ug/mL
> 30000 - - 0-10 ug/mL
TT)
0! 25000 -
sv)
20000 -
a)
U
C
15000 -
a)

g 10000 -
w
5000 -

p
2400 2600 2800
' 3000
' 3200 3400
' 3600
' 3800
' 4000
'

Control Voltage of PMT (mV)

Figure 8. Relationship between output signal of PMT and controlling voltage with FITC concentration 0.1μg/mL, 1μg/mL,
5μg/mL and 10 μg/mL
3.3 System noise and stability
Since the fluorescence was generally very weak, the system noise limited the sensitivity of the fluorometer. Phosphate
buffer solution (PBS) without fluorescence dyes was selected as samples to test the system noise with as shown in Figure
9 (PMT controlling voltage=4V, LED controlling voltage=2V). It can be seen from Figure 9(a) that the background
signal was about 0.6mV and noise (peak-peak voltage) was about 1mV.
20000
0.0014
- Detected Voltage' Intensity of fluorescence
18000
0.0012 C

111.1111i
16000
C

-NM
0.0010
S 1400 -

v 0.0008
1A
L3
p 0.0008
10000 -
-g 0.0004 Nc

w
4)
ti 0.0002 Ó 6000
o
0.0000
ó 4000

-0.0002 2000 -
c
d
0.0004
500 1000 1500 2000 2 3 4 5 6 7 8 9

Time (10ms) Times (n)

(a) Noise of the system (b) Stability of the system

Figure 9. System noise and stability test
Under the same conditions (PMT controlling voltage=4V, LED controlling voltage=2V), the stability of the system was
also tested with FITC concentration of 2 μg/mL as shown in Figure 9(b). The same samples have been detected for 9
times and the RSD was about 3% which has shown satisfied system stability.

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 3.4 Fluorescence detecting
Fluorescein isothiocyanate(FITC) is a kind of fluorescence dye with exciting wavelength 494 nm and emitting
wavelength 520nm. Therefore, selecting LSM with wavelength 494 nm and OCM with bandpass wavelength 520nm, the
fluorescence intensity was detected with FITC concentration from 10 ng/mL to 10 μg/mL as shown in Figure 10. From
Figure 10, the fluorometer in this work has shown a good linearity between fluorescence intensity and FITC
concentration with correlation coefficient of 0.98 and also shown a very high sensitivity for FITC with low concentration
of 10 ng/mL.
to'
28000
00»00Vo. N Fluorescence Signal
-o-10 ng/mL
c 24000 -I- 100 ng/mL - Allometricl Fit of B
-0-500 ng/mL
-y- 1 ug/mL
R=0.98
20000
-0- 2 ug/mL
ence Signal (Rela

ES

10 20 30 40 50 60 10' 10' 10°

Time (s) Concentration of FITC (ug /mL)

(a) Fluorescence intensity real time (b) Relationship between fluorescence intensity and FITC concentration
Figure 10. Fluorescence intensity of FITC with concentration from 10 ng/mL to 10 μg/mL

4. CONCLUSIONS
A portable and modularized fluorometer based on optical fiber has been proposed in this work. The fluorometer adopted
LEDs as light source and the LEDs were driven by a constant current source to provide stable exciting light with
different wavelength. The OCM included a modularized optical filter and used a photomultiplier tube (PMT) to detect
fluorescence signal. Via modularized design, the LSM and OCM could both selected and replaced based on different
fluorescence dyes. In order to improve the detecting dynamic range of the fluorometer, the SAM could control the light
intensity of LED source in LSM, to control the gain of PMT in OCM, and particularly, four channel signal acquisition
circuits with different gain were constructed to collect fluorescence signal simultaneously. Fluorescein isothiocyanate
(FITC) was selected as sample to test the performance of the fluorometer. The fluorometer has shown a high sensitivity
with FITC concentration of 10ng/mL and presented a good linearity from 10 ng/mL to 10 μg/mL. These results have
shown that the portable and modularized fluorometer designed in this work have good potential in fluorescence analysis.

ACKNOWLEDGEMENTS
This research project was jointly supported by the Natural Science Foundation of Shandong (Grant No. ZR2012FQ015),
the College Independent Innovation Plan of Jinan (Grant No. 201401236) and the National Training Programs of
Innovation and Entrepreneurship for Undergraduates (Grant No.201410445048).

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