Biosensors Practical session

de Maere dAertrycke, J.B., Gatt, E.

A FO-SPR based biosensor for celery

allergen detection in food
de Maere dAertrycke, J.B., Gatt, E.

Introduction
Detection of allergens in products is a great challenge for todays food sector. The presence of
allergenic compounds can be very hazardous even at very small concentrations, which makes crucial
the development of high sensitivity sensors. In this way, a biosensor, based on Fiber Optic Surface
Plasmon Resonance, were elaborated to detect celery allergen. In this practical session we work with
the FO-SPR method to detect celery using DNA as biological probe. This method is based on the
property of optical fibers that reflect the incoming light and that can create under certain conditions
an evanescence field which propagates in a direction parallel to the fiber axis. The RI around the fiber
leads to a change in the SPR signal.
In a first step, an end of the fiber optic was coated with gold by vapor deposition after removing its
cladding. A first DNA probe, complementary to a part of the celery DNA target, was then attached to
the gold coating, and another probe, complementary to the other part of the celery DNA target, was
linked to gold nanoparticles (Au NP). Therefore, when the celery DNA target is present, it should
hybridize with both the DNA sequence bound to the fiber and the one linked to the Au NP. An
increase of temperature should then cause dehybridization of the double stranded DNA, causing a
shift in SPR wavelength that could be registered by recording the light coming in the fiber optic.
In a second step, the characteristics of the sensor were evaluated. Sensitivity was determined by
analyzing solutions of increasing sucrose concentration. Then, different DNA samples were analyzed.
DNA of celery showed that the elaborated sensor responded well to the target DNA. DNA of carrot
was also analyzed to test the specificity of the sensor. Finally, measurements were made on an
unknown sample in order to determine whether it contained celery or carrot.

Results and discussion

Sensitivity measurements
Sensitivity measurement was performed by measuring the wavelength shift of surface plasmon
resonance of solutions with increasing concentration of sucrose. Table 1 shows the concentrations
of glucose that were tested. Concentration was determined both by calculation of the sucrose mass
added in a known volume of milli-Q water and measurement with a refractometer. Researches
provided the refractive index associated to the solutions tested.

Biosensors Practical session

de Maere dAertrycke, J.B., Gatt, E.

Table 1 - wavelength of SPR measured for solutions of increasing sucrose concentrations

Sucrose concentration
(mass %)
0
1.0
2.1
3.0
4.2
5.3
6.2
7.0

Refractive index

SPR (nm)

SPR (nm)

1.3330
1.3344
1.3361
1.3374
1.3391
1.3408
1.3421
1.3433

603.4611
604.9273
607.9266
610.5590
613.2832
616.0133
618.6904
622.2780

0.0000
1.4663
4.4655
7.0979
9.8221
1.5522
1.2293
18.8169

On basis of these measurements, the SPR wavelength shift response was determined. Figure 1 shows
the SPR wavelength and wavelength shift that were observed.

SPR wavelength shift [nm]

25
20
y = 1794.8x - 2393.3
R = 0.9913
15
10
5
0
1,332

1,334

1,336

1,338

1,34

1,342

1,344

refractive index

Figure 1 - wavelength shift as function of refractive index of the tested solution

The result of this experiment allowed determining the sensitivity of the sensor. Sensitivity is the ratio
of the wavelength on the increase of refractive index, which correspond to the slope of the curve
showed in figure 1. Therefore, we found that:
= 1794.8 [nm]
This graph also permitted to evaluate the limit of quantification. We decided to approximate it by the
intersection point between the calibration curve and the x-axe, since it is the point below which no
significant variation of the signal is recorded for increasing sucrose concentration. It was then
calculated as:

Biosensors Practical session

de Maere dAertrycke, J.B., Gatt, E.

Finally we observed that the linear fit hypothesis seemed to be unsuitable for sucrose concentrations
higher than 6.2%, the recorded points showing a more pronounced exponential behavior for higher
concentration. This corresponds to a refractive index of 1.3421.

Analysis of celery DNA

Here we use the functionalized fibers and NPs to determine the melting temperature (Tm) of the
specific complex formed with celery DNA. The high resolution melting analysis will allow us to
differentiate similar DNA such as the carrot DNA. Tm can be determined by calculating the melting
peak of the derivative dSPR/dT. Because the data contained a lot of noise, we followed several steps
to reduce it by using a moving average for each data point.
0
-0,1

60

70

80

90

-0,2

dSPR/dT

-0,3
-0,4
dSPR2/dT

-0,5
-0,6
-0,7
-0,8
-0,9

temperature (C)

Figure 2: dSPR/dT derivative as a function of temperature.

We did two melting cycles and in each cycle we measured two samples with two different fibers.
After taking the average result obtained with the different measurements we found this result for
the melting temperature of the complex with celery DNA:
TmCELERY = 78 3 C

The confidence interval was obtained using =0.95

Analysis of carrot DNA

Then we did the same experiment with carrot DNA to determine its Tm and be able to differentiate
celery from carrot DNA. We took the average value of our 4 values:
TmCARROT = 65.7 0.6 C
The standard deviation obtained with these four values is 0.38108 C.
The difference between the two values seems big enough to permit to discriminate the presence of
Celery DNA from that of Carrot DNA. Indeed, even with the standard deviation values there isnt any
overlap (TmCELERYMIN = 75C and TmCARROTMAX = 66.3C) between these two Tm. This statement can be
proved by a Students t-test. This test can estimate if the difference in Tm is significant by comparing

Biosensors Practical session

de Maere dAertrycke, J.B., Gatt, E.

the t-value observed and a theoretical value. In the present case, the variances are not equal, so the
t-value is given by:

The obtained t-value is then 12.134, which is higher than the theoretical value 3.182 (=0.05). We
can therefor conclude that the two melting temperature are significantly different.

Analysis of an unknown sample

Eventually we measured the Tm of the unknown sample in order to determine if it is composed of
carrot DNA or celery DNA. Once again we took the average value and obtained:
TmUNKNOWN = 64.1 0.2 C
We can clearly see that this Tm is closer to the TmCARROT and so we can say that this unknown DNA is
in fact carrot DNA.

Conclusion
In this practical session we realized a biosensor based on the properties of optic fibers. In a first part,
we established that our sensor could detect change in refractive index thanks to a change in the SPR
signal. The calculated sensitivity is 1794.8 nm. Then we could measure the melting temperature of a
double strand DNA made of a tested sequence and two probes, one of them being attached to the
fiber and the other one to an Au NP. This set up allowed to measure melting temperature, given that
when the DNA-Au NP complex melted, a change of refractive index, and of the SPR signal, could be
registered.
We were able with this device to discriminate quite accurately different but close DNA sequences on
basis of their respective Tm. In this experiment the selectivity for celery DNA was successfully tested
against carrot DNA, Tm being respectively 75C and 66.3 C. The measurements of those Tm
permitted us in a third experiment to identify an unknown sample as composed of carrot DNA.
This method is quite interesting for specific detection of allergens in the processed food products,
which can be quite complicated because of the similarities in term of composition of many products.
Still, the identification of the tested DNA was simple because the sample contained only one type of
DNA, either carrot or celery. In case of a mixed food matrix the analysis would be much more
complex. Indeed celery DNA could bind as well as others non-targeted DNA sequences and another
signal, resulting in the combination of melting DNAs at different temperature would be registered,
leading to more complex discrimination. Moreover this method needs to extract the DNA from the
sample which is not always easy and could lead to loss of interesting DNA sequences.

Biosensors Practical session

de Maere dAertrycke, J.B., Gatt, E.

Two previously elaborated biosensors for the detection of allergens

The sensor developed below is certainly not the first attempt to detect celery allergens. In this part,
we will expose two other strategies that have been experimented to detect it in processed food. The
first sensor, elaborated by H. Wang et al. (2011), is an ELISA test specific to the allergenic protein
component Api g 1.01. The second one, made by M. Fuchs et al. (2012), explores the use of real-time
PCR method to detect the presence of celery in food.
The ELISA-based sensor required two different monoclonal antibodies that were obtained by
infecting mice with Api g 1.01 and collecting spleen cells to fuse them with a myeloma line and form
hybridoma. These hybridoma were then screened to select the two ones that produce monoclonal
antibodies with the best properties for the ELISA test, such as low cross-reactivity or high affinity. The
ELISA test was based on sandwich configuration: a first antibody was used to coat the microtiter
plate. It was then incubated first with the tested sample, then with a blocking buffer, and finally with
the second antibody, labelled with HRP. Luminol, the substrate of HRP, was finally added, its
oxidation, emitting a characteristic light, could then be recorded by a spectrophotometer.
The second biosensor is based on real-time PCR. DNA is extracted from the tested sample and is
mixed with PCR reactants containing four primers specific for the amplification of two different
celery DNA sequences. A probe is also added, composed of a specific DNA sequence at which a
fluorescent reporter and a quencher are bound at the two ends. Hybridization of the probe induces
physical separation of the reporter and the quencher, which causes the emission of a fluorescent
signal, proportional to the number of DNA sequences amplified.
A great advantage of the first technique is that it doesnt require too complex purification step, while
this is clearly a drawback for the second technique. Indeed, it appears that performance and results
of the DNA-based method is strongly dependent on the protocol applied to extract DNA. However,
the first method shows a lower limit of detection (95 pg/L versus 2pg celery DNA/L).

Sources
Fuchs, M., Cuchna-Markl, M.,Hochegger, R. (2012). Development and validation of a novel real-time
PCR method for the detection of celery (Apium graveolens) in food. Food Chemistry, 130, 189-195
Wang, H., Li, G., Yuan, F., Wu, Y., Chen, Y. (2011). Detection of the allergenic celery protein
component (Api g 1.01) in foods by immunoassay. Eur. Food Res. Technol. 233, 1023-1028