- Email: [email protected]
A Biosensor to Measure Fungicide Iprovalicarb Residues in Agricultural Products
EAEF 1(2) : 63-67, 2008
Invited Paper
A Biosensor to Measure Fungicide Iprovalicarb Residues in Agricultural Products* Han Keun CHO *1, Kee Sung KYUNG*2 Abstract Using an enzyme-linked immunosorbent assay (ELISA), a biosensor was developed to rapidly measure fungicide iprovalicarb residues in agricultural products. The biosensor was designed to include micro-pumps and solenoid valves for fluid transport, a spectrophotometer cuvet as a reaction chamber, a photodiode with a light-emitting diode for optical density measurement, and a control microcomputer to implement assay. The rate of change in optical density of the cuvette was read as final signal output.
Micro-pumps were evaluated to investigate their
delivery capability. The maximum error and the coefficient of variation were found to be 4.3% and 4.6%, respectively. To predict the concentration of the iprovalicarb residue in agricultural products, a linear calibration model was obtained with r-square values of 0.992 for potato and 0.985 for onion.
In validation test for the samples
of potatoes and onions against the high performance liquid chromatography (HPLC), very high correlation values were obtained as 0.996 and 0.993, respectively. Using the cuvette immobilized with antigen, it took 21-minutes for the biosensor to complete the measuring process of the iprovalicarb residues . The measurement assay was successfully automated with an adequate sensitivity level to measure the government limits for iprovalicarb residues. [Keywords] biosensor, elisa, antibody, antigen, iprovalicarb, fungicide residues
I
Introduction
The fungicide iprovalicarb is extensively used in Korea for the control of anthracnose, downy mildew and black shank in many crops such as potato, onion, cucumber, tomato, grape, and watermelon. Although its half-life is not very long, residue problem may arise in agriculturalG products and environment. Iprovalicarb, isopropyl 2-methyl-1-[(1-p-tolylethyl) carba moyl]-(S)-propylcarbamate, belongs to the new amino acid amidocarbamate class of compounds and is a protective, curative, and eradicative systemic fungicide introduced by Bayer AG in 1999. The product is a mixture of (SS)-and (SR)-diastereoisomers (Lee et al., 2004).
iprovalicarb residues in agricultural products. A biosensor in general can be defined as an analytical device consisting of a biological sensing element in close association with a physical transducer.
The biological recognition element performs the
molecular recognition function responsible to specific sensing of an analyte of interest and then the physical transducer converts the recognition event into a measurable optical or electrical signal. The objective of this research was to (1) design an automated biosensorGto implement an enzyme immunoassay for rapid detection of the fungicide iprovalicarb residues in vegetables, and (2) calibrate the biosensor and evaluate its measurement performance.
A simple, sensitive, and cost-effective ELISA was developed by Lee et al. (2004) for the detection of iprovalicarb residue in agricultural and environment samples. Automated biosensor systems are needed at both the producer and wholesaler levels to indicate the presence of the
IIG Materials and Methods 1. Chemicals Iprovalicarb and p-methylphenylamine, of analytical grade were gifts from BayerG AG.
BovinG serum albumin (BSA),
* Partly presented at the Conference of ISMAB in May 2008 *1 KSAM Member, Corresponding author, Professor, Department of Biosystems Engineering, Chungbuk National University, Cheongju, 361-763, Rep. of Korea; phone: +82-43-261-2585, fax: +82-43-271-4413; [email protected] *2 Assistant Professor, Department of Agricultural Chemisitry, Chungbuk National University, Cheongju, 361-763, Rep. of Korea.
64
Engineering in Agriculture, Environment and Food Vol. 1, No. 2 (2008)
obtained from Sigma Chemical Co. (St. Louis, MO). A
solenoid activated pumps, each rated at 50 LG per stroke (LPLAG1220050L, The Lee Co., Westbrook, Ct.). Two-way
polyclonal antibody produced against a hapten conjugated and
normally-closed valves were used for priming the lines
goat anti-rabitG (IgG) and peroxidaseG conjugate, were all
a coating antigen were provided by Lee et al. (2004). Buffer
(LFFAG 1201710H and LFFAG 1201410H, The Lee Co.). A
solutions were normal strength phosphate-buffered saline (1
separate pump and lines were used for the labeled
โPBS, 0.001 M KH2PO4, pH7.5), PBST(1โPBS containing
iprovalicarbG conjugate to minimize contamination of the
0.05% Tween 20), carbonate buffer(pH 9.6), and 0.05 M
reagent pump, manifold, and priming valve with peroxidase.
borate buffer (pH 8). Each buffer was prepared according to
The mix pump was used to provide recirculation of the
the method used in Lee et al. (2004).
reaction chamber to enhance the binding process and the air pump was used to dry out the tubes. The substrate, conjugate, mix, and supply lines to the
2. Assay development The previous research developed an enzyme immunoassay for
reaction chamber were made from 1.6 mm inner diameter
iprovalicarb suitable for use in a sensor by modifying the
VitonG tubing (06434-01, Cole-ParmerG Instrument Corp.,
results of Cho et al. (2006). The dilution of coating antigen
Vernon Hills, IL.), with the remaining lines TygonG tubing
and both primary and secondary antibodies were increased to
(14-169-1B, Fisher Scientific, Pittsburgh, Pa) of the same
improve the competition and incubation time was reduced to
inner diameter. Stainless steel tubes with an inner diameterGof
save test time.
1.1 mm (HTX-17, Small Parts Inc., Miami Lakes, CA.) were
The modified procedure for iprovalicarb
ELISAGis summarized in Table 1.
inserted in a TeflonGspacer cap and used to inject the reagents and drain the chamber. Absorbance of the substrate was measured by positioning a 645 nmGlight-emitting diode (LED,
Table 1 Protocol for the ELISA used in the biosensor (Cho et al.,
HLMP-4101, Hewlett Packard, San Jose, CA) on one side of the reaction chamber and a large-area photodiodeG(BPXG61,
2006) Immobilization of Antigen (By hand) 1. Immobilize antigen on cuvette (1 mL, 1 Ǵg/mL, 12 hr, 4͠) 2. Washing cuvette (1PBS + 0.05% Tween20, 5 times) 3. Blocking cuvette (2 mL, 1PBS + 3% skim milk, 1 hr, 37͠) 4. Washing cuvette (1PBS + 0.05% Tween20, 5 times) Binding (By the biosensor) 5. Add 500 ǴL of sample (5000, 200, 8, 0.32, 0.0128 ng/mL) 6. Add 500 ǴL of the 1st-antibody (1:4,000) 7. Incubate for 5 or 3 minutes at 24͠ 8. Washing cuvette (1PBS + 0.05% Tween20, 5 times) 9. Add 1 mL of the 2nd-antibody (1:1,000) 10. Incubate for 5 or 3 minutes at 24͠
Siemens Components, Inc., Cupertino, CA) on the other side. Light from the LED was collimated with a simple peep-hole configuration (DelwichGet al., 2001). Light current from the photodiodeGwas fed to the input of a photovoltaic amplifier with a gain of 104G V/A.
frequency noise was then removed with a 4th order ButterworthGlow-pass filter (fc=10 Hz). An inverter with a fixed gain of 10 and adjustable offset was used in the final stage, with output voltage proportional to the intensity of light transmitted through the substrate. Relative optical density was calculated based on the output at the start of substrate development.
11. Washing cuvette (1PBS + 0.05% Tween20, 5 times)
Air
Development (By the biosensor)
Wash Sample 1st Ab Substrate solution
12. Add 1 mL of substrate buffer
Priming valve (3-way)
(citrate buffer, 0.6% TMB, 1% H2O2 ) 13. Incubate for 5 minutes at 24͠ 14. Read optical density
Low
Reagent valves (2-way)
Manifold
Reagent pump
Drain pump
Waste nd
2 Antibody pump
Mix pump Stainless steel tubes
3. Sensor design A schematic of the fluid handling system is shown in figure 1. A standard quartz cuvetteG(TypeG5 H, NSGGPrecision Cells,
LED
Photodiode 2nd Antibody Reaction chamber
Inc., Farmingdale, N.Y.) was used as the reaction chamber to minimize optical and chemical interfaces. Reagents were pumped into and out of the reaction chamber with four
Fig. 1
Schematic of the biosensor fluidics
65
CHO, KYUNG : A Biosensor to Detect Fungicide Iprovalicarb in Vegetebles 4. Prototype of the biosensor A prototype of the biosensor developed is shown in Figure
Table 2 Control sequence for the biosensor
2. Two control circuit boards are located in the upper side of
Binding cycle
the sensor box, a larger board (1) in rear includes a
كSample valve (500 ǴL), Reagent pump (10 strokes)
microcomputer, signal processing and control part, a smaller board (2) in front includes a LCD (liquid cristal display) to
ك1st antibody valve (500 ǴL), Reagent pump (10 strokes)
display the final concentration of the sample and five control
كMix pump (28 strokes)
switches. Six solenoid valves (3) and four micro-pumps (4)
كIncubate 5 or 3 min. (24͠)
are located in middle part of the sensor box. Four chemical
Washing cycle 5
vessels (5) are located at the left lower side and a black
كDrain cuvet
reaction chamber (6) with absorbance measuring unit is
كWashing valve (1.2 mL), Reagent pump (26 strokes)
located at the right lower side of the sensor box.
كMix pump (28 strokes) Labeling cycle ك2nd antibody pump (19 strokes) كIncubate 5 or 3 min. (24͠) Washing cycle 5 Development cycle كSubstrate valve (1 mL), Reagent pump (17 strokes) كIncubate 5 min. (24͠) كCalculate OD rate (10 s) Cleaning cycle كClear sample valve G
كClear substrate valve كAdd washing solution
Fig. 2
Picture of the biosensor prototype 6. Evaluation of the biosensor The performance of fluid transfer by pumps and valves was
5. Control of the biosensor Figure 3 shows the control logic diagram of the biosensor.
investigated using distilled water and an electronic weigh
A microcomputer (AVR, ATmega 8535, ATMEL) was used
scale. Each transfer process was replicated 10 times. The
to sequence the pumps and valves with Darlington driver, and
calibration curve of the biosensor was obtained in two ways:
read the sensor output with A/D conversion. Finally the
the general calibration curve with iprovalicarb samples in
concentration of the iprovalicarb was displayed as voltage
PBS buffer which concentration is known and the specific
output on the LCD. The detail control sequence of the
calibration curve according to the agricultural products such
biosensor is summarized in Table 2.
as
cucumbers
and
potatoes.
Finally
the
biosensor
measurements were compared against HPLC (HP 1100 series, Agilent Technologies Korea, Young-In Scientific, Korea) Air
Darlington driver for valves
Photodiode Photodiode
Wash Sample
1st Antibody
Signal Signal processor processor
Microcontroller
Substrate
Priming (3-way)
LED LED
LCD LCD
Darlington driver for pumps
As the buffer standard samples, five levels of concentration prepared are 0.0128, 0.32, 8, 200, and 5000 ng/mL. The dilution ratios of antibody used are 1:2,000 for the 1st antibody and 1:500 for the 2nd antibody. The incubation time was varied from 15 minutes to 11 minutes. Onion and potato samples were used for evaluation of the
Reagent
biosensor.
Drain
sample has been described in more detail (Lee et al., 2004).
2nd Antibody Mixing
Fig. 3
results.
Control logic of the biosensor
The preparation procedure of extracting each
The potato samples were fortified with the stock solution of iprovalicarb dissolved in CH3CN to 1, 2, 3 ng/mL and the onion samples were fortified to 2, 4, 6 ng/mL.
66
Engineering in Agriculture, Environment and Food Vol. 1, No. 2 (2008)
III Results and Discussion
y
1. Chemical transfer performance The result of dispensed volume to a cuvette by
a b b 1 ( x / c) d
(1)
where y : Optical density
micro-pumps is shown in Table 3. In case of reagent pumps,
x : concentration (ng/mL) a : initial y value b : final y value
the error rates ranged from 0.7 to 2.1 %. The error rates of the 2nd antibody pump, mixing pump and drain pump are shown to be 4.3%, 1.6% and 3.3%, respectively. The coefficients
c : center d : power
of variation (CV) ranged from 0.1 to 4.6%. All pumps were found to be acceptable to transfer chemicals accurately. Table 3
Test results of dispensed volume to a cuvette
Table 4
by micro-pumps
calibration curve
Set
Meas’d
Error
CV
Incubation time, minutes
(mL)
(mL)
(%)
(%)
(Bind-label-development)
Sample
500.0
510.5
2.1
0.5
1st Ab
500.0
507.8
1.6
1.5
Substrate
1000.0
1019.2
1.9
0.3
Wash
1200.0
1208.2
0.7
0.3
Micro-pumps
Reagent
Parameter values of sigmoidal model for
nd
2 Ab
1000.0
1042.7
4.3
4.6
Mixing
1200.0
1219.6
1.6
1.6
Drain
1200.0
1239.8
3.3
0.1
Sigmoidal model parameters a
b
c
d
5-5-5
0.202 0.055 18.609 0.311
3-3-5
0.552 -0.349 68.477 0.069
3. Calibration with crops Results from calibrating the biosensor with known samples of potato and onion are plotted in Figure 5. The horizontal axis shows the concentration of the vegetable extract and the vertical axis shows the relative optical
2. Calibration with buffer solution In general, the similar magnitudes of optical density for the
density. The calibration equation was derived by solving
sensor were observed as compared with results obtained when
the regression result for the predicted concentration.
In the
performing the assay by hand.
Figure 4 shows the
concentration range tested from 0.0128 to 5000 ng/mL, the
calibration curve with standard sample solution of the
optical density showed a good linear correlation with
iprovalicarb for different incubation times. The horizontal
iprovalicarb concentration. The r-square values of the linear
axis shows the concentration of standard iprovalicarb solution
models are obtained as 0.985 and 0.988 for potato and onion,
and the vertical axis shows the relative optical density. The
respectively. The linear regression equations of both onion
r-square value of the sigmoide model is 0.999 and 0.997 for
and potato are shown in the plot.
each incubation time. The model equation is shown in Eq. (1) and those parameter values of sigmoid model are shown in Table 4. 0.28 Onion Potato
0.26 0.24
Incubation 5-5-5 min Incubation 3-3-5 min
0.20 0.18
Optical density
0.16 0.14
Optical density
0.22
0.22
y=0.206-0.032x
0.20
r-sq=0.985
0.18 0.16 0.14
y=0.189-0.025x
0.12
0.12
r-sq=0.988
0.10
0.10
0.08
0.08 0.01
0.06
0.1
1
10
100
1000
10000
Iprovalicarb concentration, ng/mL
0.04 0.01
0.1
1
10
100
1000
10000
Iprovalicarb concentration, ng/mL
Fig. 5
Sensor calibration results for onion and potato extracts
Fig. 4
Sensor calibration results for the incubation time
67
CHO, KYUNG : A Biosensor to Detect Fungicide Iprovalicarb in Vegetebles 4. Comparison of the biosensor with HPLC As illustrated in Figures 6 and 7, a good linear correlation is shown for the biosensor measurements at the iprovalicarb concentration tested with HPLC measurements.
As shown
in the figures the correlation coefficients were 0.996 and 0.993 for the potato and the onion, respectively.
These
results suggested that the fungicide iprovalicarb residue in potato and onion could be measured with developed biosensor in the comparable degree of HPLC accuracy.
V Summary and Conclusions A
biosensorG for
rapid
measurement
of
fungicide
iprovalicarbG residue was designed and constructed. An enzyme immunoassay developed by Lee et al. (2004) and modified by Cho et al. (2006) was automated.
This
biosensorGincludes a reaction chamber with absorbance sensor, micro fluidics, signal conditioner, and a microcomputer for control. Results from the calibration test showed that the iprovalicarbGresidues in potato and onion could be measured with the degree of accuracy as HPLC.
The sensitivity level
is also adequate to detect the government limits for iprovalicarb residues indicating that the measurement assay
3.5
Concentration by biosensor, ng/mL
y=1.148x-0.114 (r=0.996)
was successfully implemented.
3.0
This biosensor could
execute a measurement cycle in 21-minutes with the cuvette 2.5
immobilized.
2.0
References
1.5
Cho, H. K., K. S. Kyung and E. Y. Lee. 2006. Enzyme immunoassay
1.0
for rapid detection of the fungicide iprovalicarb residues. J. of
0.5
Biosystems Eng. 31(6):535-540. (In Korean) 0.0
Crowther, J. R. 2001. The ELISA Guidebook. Hummana Press, 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Concentration by HPLC, ng/mL
Totowa, New Jersey. Delwiche, M. Tang, X. BonDurant, R. Munro, C. 2001. Improved Biosensor for measurement of Progesterone in Bovine milk. Trans. of the ASAE 44(6):1997-2002.
Fig. 6
Correlation of potato iprovalicarb residues measured by the biosensor and HPLC.
Lee, J. K., S. H. Park, O. S. Park, E. Y. Lee, Y. J. Kim and K. S. Kyung. 2004. Development of an ELISA for the detection of the residues of the fungicide iprovalicarb. J. Agric. Food Chem. 52: 6680-6686. (Accepted : 31. July. 2008)
y=1.039x-0.186 (r=0.993)
Concentration by biosensor, ng/mL
6
5
4
3
2
1
0 0
1
2
3
4
5
6
Concentration by HPLC, ng/mL
Fig. 7
Correlation of onion iprovalicarb residues measured by the biosensor and HPLC.
Invited Paper
A Biosensor to Measure Fungicide Iprovalicarb Residues in Agricultural Products* Han Keun CHO *1, Kee Sung KYUNG*2 Abstract Using an enzyme-linked immunosorbent assay (ELISA), a biosensor was developed to rapidly measure fungicide iprovalicarb residues in agricultural products. The biosensor was designed to include micro-pumps and solenoid valves for fluid transport, a spectrophotometer cuvet as a reaction chamber, a photodiode with a light-emitting diode for optical density measurement, and a control microcomputer to implement assay. The rate of change in optical density of the cuvette was read as final signal output.
Micro-pumps were evaluated to investigate their
delivery capability. The maximum error and the coefficient of variation were found to be 4.3% and 4.6%, respectively. To predict the concentration of the iprovalicarb residue in agricultural products, a linear calibration model was obtained with r-square values of 0.992 for potato and 0.985 for onion.
In validation test for the samples
of potatoes and onions against the high performance liquid chromatography (HPLC), very high correlation values were obtained as 0.996 and 0.993, respectively. Using the cuvette immobilized with antigen, it took 21-minutes for the biosensor to complete the measuring process of the iprovalicarb residues . The measurement assay was successfully automated with an adequate sensitivity level to measure the government limits for iprovalicarb residues. [Keywords] biosensor, elisa, antibody, antigen, iprovalicarb, fungicide residues
I
Introduction
The fungicide iprovalicarb is extensively used in Korea for the control of anthracnose, downy mildew and black shank in many crops such as potato, onion, cucumber, tomato, grape, and watermelon. Although its half-life is not very long, residue problem may arise in agriculturalG products and environment. Iprovalicarb, isopropyl 2-methyl-1-[(1-p-tolylethyl) carba moyl]-(S)-propylcarbamate, belongs to the new amino acid amidocarbamate class of compounds and is a protective, curative, and eradicative systemic fungicide introduced by Bayer AG in 1999. The product is a mixture of (SS)-and (SR)-diastereoisomers (Lee et al., 2004).
iprovalicarb residues in agricultural products. A biosensor in general can be defined as an analytical device consisting of a biological sensing element in close association with a physical transducer.
The biological recognition element performs the
molecular recognition function responsible to specific sensing of an analyte of interest and then the physical transducer converts the recognition event into a measurable optical or electrical signal. The objective of this research was to (1) design an automated biosensorGto implement an enzyme immunoassay for rapid detection of the fungicide iprovalicarb residues in vegetables, and (2) calibrate the biosensor and evaluate its measurement performance.
A simple, sensitive, and cost-effective ELISA was developed by Lee et al. (2004) for the detection of iprovalicarb residue in agricultural and environment samples. Automated biosensor systems are needed at both the producer and wholesaler levels to indicate the presence of the
IIG Materials and Methods 1. Chemicals Iprovalicarb and p-methylphenylamine, of analytical grade were gifts from BayerG AG.
BovinG serum albumin (BSA),
* Partly presented at the Conference of ISMAB in May 2008 *1 KSAM Member, Corresponding author, Professor, Department of Biosystems Engineering, Chungbuk National University, Cheongju, 361-763, Rep. of Korea; phone: +82-43-261-2585, fax: +82-43-271-4413; [email protected] *2 Assistant Professor, Department of Agricultural Chemisitry, Chungbuk National University, Cheongju, 361-763, Rep. of Korea.
64
Engineering in Agriculture, Environment and Food Vol. 1, No. 2 (2008)
obtained from Sigma Chemical Co. (St. Louis, MO). A
solenoid activated pumps, each rated at 50 LG per stroke (LPLAG1220050L, The Lee Co., Westbrook, Ct.). Two-way
polyclonal antibody produced against a hapten conjugated and
normally-closed valves were used for priming the lines
goat anti-rabitG (IgG) and peroxidaseG conjugate, were all
a coating antigen were provided by Lee et al. (2004). Buffer
(LFFAG 1201710H and LFFAG 1201410H, The Lee Co.). A
solutions were normal strength phosphate-buffered saline (1
separate pump and lines were used for the labeled
โPBS, 0.001 M KH2PO4, pH7.5), PBST(1โPBS containing
iprovalicarbG conjugate to minimize contamination of the
0.05% Tween 20), carbonate buffer(pH 9.6), and 0.05 M
reagent pump, manifold, and priming valve with peroxidase.
borate buffer (pH 8). Each buffer was prepared according to
The mix pump was used to provide recirculation of the
the method used in Lee et al. (2004).
reaction chamber to enhance the binding process and the air pump was used to dry out the tubes. The substrate, conjugate, mix, and supply lines to the
2. Assay development The previous research developed an enzyme immunoassay for
reaction chamber were made from 1.6 mm inner diameter
iprovalicarb suitable for use in a sensor by modifying the
VitonG tubing (06434-01, Cole-ParmerG Instrument Corp.,
results of Cho et al. (2006). The dilution of coating antigen
Vernon Hills, IL.), with the remaining lines TygonG tubing
and both primary and secondary antibodies were increased to
(14-169-1B, Fisher Scientific, Pittsburgh, Pa) of the same
improve the competition and incubation time was reduced to
inner diameter. Stainless steel tubes with an inner diameterGof
save test time.
1.1 mm (HTX-17, Small Parts Inc., Miami Lakes, CA.) were
The modified procedure for iprovalicarb
ELISAGis summarized in Table 1.
inserted in a TeflonGspacer cap and used to inject the reagents and drain the chamber. Absorbance of the substrate was measured by positioning a 645 nmGlight-emitting diode (LED,
Table 1 Protocol for the ELISA used in the biosensor (Cho et al.,
HLMP-4101, Hewlett Packard, San Jose, CA) on one side of the reaction chamber and a large-area photodiodeG(BPXG61,
2006) Immobilization of Antigen (By hand) 1. Immobilize antigen on cuvette (1 mL, 1 Ǵg/mL, 12 hr, 4͠) 2. Washing cuvette (1PBS + 0.05% Tween20, 5 times) 3. Blocking cuvette (2 mL, 1PBS + 3% skim milk, 1 hr, 37͠) 4. Washing cuvette (1PBS + 0.05% Tween20, 5 times) Binding (By the biosensor) 5. Add 500 ǴL of sample (5000, 200, 8, 0.32, 0.0128 ng/mL) 6. Add 500 ǴL of the 1st-antibody (1:4,000) 7. Incubate for 5 or 3 minutes at 24͠ 8. Washing cuvette (1PBS + 0.05% Tween20, 5 times) 9. Add 1 mL of the 2nd-antibody (1:1,000) 10. Incubate for 5 or 3 minutes at 24͠
Siemens Components, Inc., Cupertino, CA) on the other side. Light from the LED was collimated with a simple peep-hole configuration (DelwichGet al., 2001). Light current from the photodiodeGwas fed to the input of a photovoltaic amplifier with a gain of 104G V/A.
frequency noise was then removed with a 4th order ButterworthGlow-pass filter (fc=10 Hz). An inverter with a fixed gain of 10 and adjustable offset was used in the final stage, with output voltage proportional to the intensity of light transmitted through the substrate. Relative optical density was calculated based on the output at the start of substrate development.
11. Washing cuvette (1PBS + 0.05% Tween20, 5 times)
Air
Development (By the biosensor)
Wash Sample 1st Ab Substrate solution
12. Add 1 mL of substrate buffer
Priming valve (3-way)
(citrate buffer, 0.6% TMB, 1% H2O2 ) 13. Incubate for 5 minutes at 24͠ 14. Read optical density
Low
Reagent valves (2-way)
Manifold
Reagent pump
Drain pump
Waste nd
2 Antibody pump
Mix pump Stainless steel tubes
3. Sensor design A schematic of the fluid handling system is shown in figure 1. A standard quartz cuvetteG(TypeG5 H, NSGGPrecision Cells,
LED
Photodiode 2nd Antibody Reaction chamber
Inc., Farmingdale, N.Y.) was used as the reaction chamber to minimize optical and chemical interfaces. Reagents were pumped into and out of the reaction chamber with four
Fig. 1
Schematic of the biosensor fluidics
65
CHO, KYUNG : A Biosensor to Detect Fungicide Iprovalicarb in Vegetebles 4. Prototype of the biosensor A prototype of the biosensor developed is shown in Figure
Table 2 Control sequence for the biosensor
2. Two control circuit boards are located in the upper side of
Binding cycle
the sensor box, a larger board (1) in rear includes a
كSample valve (500 ǴL), Reagent pump (10 strokes)
microcomputer, signal processing and control part, a smaller board (2) in front includes a LCD (liquid cristal display) to
ك1st antibody valve (500 ǴL), Reagent pump (10 strokes)
display the final concentration of the sample and five control
كMix pump (28 strokes)
switches. Six solenoid valves (3) and four micro-pumps (4)
كIncubate 5 or 3 min. (24͠)
are located in middle part of the sensor box. Four chemical
Washing cycle 5
vessels (5) are located at the left lower side and a black
كDrain cuvet
reaction chamber (6) with absorbance measuring unit is
كWashing valve (1.2 mL), Reagent pump (26 strokes)
located at the right lower side of the sensor box.
كMix pump (28 strokes) Labeling cycle ك2nd antibody pump (19 strokes) كIncubate 5 or 3 min. (24͠) Washing cycle 5 Development cycle كSubstrate valve (1 mL), Reagent pump (17 strokes) كIncubate 5 min. (24͠) كCalculate OD rate (10 s) Cleaning cycle كClear sample valve G
كClear substrate valve كAdd washing solution
Fig. 2
Picture of the biosensor prototype 6. Evaluation of the biosensor The performance of fluid transfer by pumps and valves was
5. Control of the biosensor Figure 3 shows the control logic diagram of the biosensor.
investigated using distilled water and an electronic weigh
A microcomputer (AVR, ATmega 8535, ATMEL) was used
scale. Each transfer process was replicated 10 times. The
to sequence the pumps and valves with Darlington driver, and
calibration curve of the biosensor was obtained in two ways:
read the sensor output with A/D conversion. Finally the
the general calibration curve with iprovalicarb samples in
concentration of the iprovalicarb was displayed as voltage
PBS buffer which concentration is known and the specific
output on the LCD. The detail control sequence of the
calibration curve according to the agricultural products such
biosensor is summarized in Table 2.
as
cucumbers
and
potatoes.
Finally
the
biosensor
measurements were compared against HPLC (HP 1100 series, Agilent Technologies Korea, Young-In Scientific, Korea) Air
Darlington driver for valves
Photodiode Photodiode
Wash Sample
1st Antibody
Signal Signal processor processor
Microcontroller
Substrate
Priming (3-way)
LED LED
LCD LCD
Darlington driver for pumps
As the buffer standard samples, five levels of concentration prepared are 0.0128, 0.32, 8, 200, and 5000 ng/mL. The dilution ratios of antibody used are 1:2,000 for the 1st antibody and 1:500 for the 2nd antibody. The incubation time was varied from 15 minutes to 11 minutes. Onion and potato samples were used for evaluation of the
Reagent
biosensor.
Drain
sample has been described in more detail (Lee et al., 2004).
2nd Antibody Mixing
Fig. 3
results.
Control logic of the biosensor
The preparation procedure of extracting each
The potato samples were fortified with the stock solution of iprovalicarb dissolved in CH3CN to 1, 2, 3 ng/mL and the onion samples were fortified to 2, 4, 6 ng/mL.
66
Engineering in Agriculture, Environment and Food Vol. 1, No. 2 (2008)
III Results and Discussion
y
1. Chemical transfer performance The result of dispensed volume to a cuvette by
a b b 1 ( x / c) d
(1)
where y : Optical density
micro-pumps is shown in Table 3. In case of reagent pumps,
x : concentration (ng/mL) a : initial y value b : final y value
the error rates ranged from 0.7 to 2.1 %. The error rates of the 2nd antibody pump, mixing pump and drain pump are shown to be 4.3%, 1.6% and 3.3%, respectively. The coefficients
c : center d : power
of variation (CV) ranged from 0.1 to 4.6%. All pumps were found to be acceptable to transfer chemicals accurately. Table 3
Test results of dispensed volume to a cuvette
Table 4
by micro-pumps
calibration curve
Set
Meas’d
Error
CV
Incubation time, minutes
(mL)
(mL)
(%)
(%)
(Bind-label-development)
Sample
500.0
510.5
2.1
0.5
1st Ab
500.0
507.8
1.6
1.5
Substrate
1000.0
1019.2
1.9
0.3
Wash
1200.0
1208.2
0.7
0.3
Micro-pumps
Reagent
Parameter values of sigmoidal model for
nd
2 Ab
1000.0
1042.7
4.3
4.6
Mixing
1200.0
1219.6
1.6
1.6
Drain
1200.0
1239.8
3.3
0.1
Sigmoidal model parameters a
b
c
d
5-5-5
0.202 0.055 18.609 0.311
3-3-5
0.552 -0.349 68.477 0.069
3. Calibration with crops Results from calibrating the biosensor with known samples of potato and onion are plotted in Figure 5. The horizontal axis shows the concentration of the vegetable extract and the vertical axis shows the relative optical
2. Calibration with buffer solution In general, the similar magnitudes of optical density for the
density. The calibration equation was derived by solving
sensor were observed as compared with results obtained when
the regression result for the predicted concentration.
In the
performing the assay by hand.
Figure 4 shows the
concentration range tested from 0.0128 to 5000 ng/mL, the
calibration curve with standard sample solution of the
optical density showed a good linear correlation with
iprovalicarb for different incubation times. The horizontal
iprovalicarb concentration. The r-square values of the linear
axis shows the concentration of standard iprovalicarb solution
models are obtained as 0.985 and 0.988 for potato and onion,
and the vertical axis shows the relative optical density. The
respectively. The linear regression equations of both onion
r-square value of the sigmoide model is 0.999 and 0.997 for
and potato are shown in the plot.
each incubation time. The model equation is shown in Eq. (1) and those parameter values of sigmoid model are shown in Table 4. 0.28 Onion Potato
0.26 0.24
Incubation 5-5-5 min Incubation 3-3-5 min
0.20 0.18
Optical density
0.16 0.14
Optical density
0.22
0.22
y=0.206-0.032x
0.20
r-sq=0.985
0.18 0.16 0.14
y=0.189-0.025x
0.12
0.12
r-sq=0.988
0.10
0.10
0.08
0.08 0.01
0.06
0.1
1
10
100
1000
10000
Iprovalicarb concentration, ng/mL
0.04 0.01
0.1
1
10
100
1000
10000
Iprovalicarb concentration, ng/mL
Fig. 5
Sensor calibration results for onion and potato extracts
Fig. 4
Sensor calibration results for the incubation time
67
CHO, KYUNG : A Biosensor to Detect Fungicide Iprovalicarb in Vegetebles 4. Comparison of the biosensor with HPLC As illustrated in Figures 6 and 7, a good linear correlation is shown for the biosensor measurements at the iprovalicarb concentration tested with HPLC measurements.
As shown
in the figures the correlation coefficients were 0.996 and 0.993 for the potato and the onion, respectively.
These
results suggested that the fungicide iprovalicarb residue in potato and onion could be measured with developed biosensor in the comparable degree of HPLC accuracy.
V Summary and Conclusions A
biosensorG for
rapid
measurement
of
fungicide
iprovalicarbG residue was designed and constructed. An enzyme immunoassay developed by Lee et al. (2004) and modified by Cho et al. (2006) was automated.
This
biosensorGincludes a reaction chamber with absorbance sensor, micro fluidics, signal conditioner, and a microcomputer for control. Results from the calibration test showed that the iprovalicarbGresidues in potato and onion could be measured with the degree of accuracy as HPLC.
The sensitivity level
is also adequate to detect the government limits for iprovalicarb residues indicating that the measurement assay
3.5
Concentration by biosensor, ng/mL
y=1.148x-0.114 (r=0.996)
was successfully implemented.
3.0
This biosensor could
execute a measurement cycle in 21-minutes with the cuvette 2.5
immobilized.
2.0
References
1.5
Cho, H. K., K. S. Kyung and E. Y. Lee. 2006. Enzyme immunoassay
1.0
for rapid detection of the fungicide iprovalicarb residues. J. of
0.5
Biosystems Eng. 31(6):535-540. (In Korean) 0.0
Crowther, J. R. 2001. The ELISA Guidebook. Hummana Press, 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Concentration by HPLC, ng/mL
Totowa, New Jersey. Delwiche, M. Tang, X. BonDurant, R. Munro, C. 2001. Improved Biosensor for measurement of Progesterone in Bovine milk. Trans. of the ASAE 44(6):1997-2002.
Fig. 6
Correlation of potato iprovalicarb residues measured by the biosensor and HPLC.
Lee, J. K., S. H. Park, O. S. Park, E. Y. Lee, Y. J. Kim and K. S. Kyung. 2004. Development of an ELISA for the detection of the residues of the fungicide iprovalicarb. J. Agric. Food Chem. 52: 6680-6686. (Accepted : 31. July. 2008)
y=1.039x-0.186 (r=0.993)
Concentration by biosensor, ng/mL
6
5
4
3
2
1
0 0
1
2
3
4
5
6
Concentration by HPLC, ng/mL
Fig. 7
Correlation of onion iprovalicarb residues measured by the biosensor and HPLC.