Biosensor-Based Determination of Riboflavin in Milk Samples

Article abstract of DOI:10.1021/ac034876a

An assay for quantification of riboflavin (Rf) in milk-based products has been developed using the principle of surface plasmon resonance with on-chip measurement. The quantification was done indirectly by measuring excess of Rf binding protein (RBP) that remains free after complexation with Rf molecules originally present in the sample solution. The chip was modified with covalently immobilized Rf in order to bind the RBP in excess. A chemical modification was performed to introduce a reactive ester group at the N-3 position of the natural Rf to bind amino groups present on the chip surface. Calibration solutions were prepared by mixing a range of Rf standard solutions with an optimized concentration of RBP. The Rf content in the milk-based products was then measured by comparison of the response against the calibration. Results obtained were very close to those from an official HPLC-fluorescence procedure. The limit of quantification was determined to 234 μg/L and the limit of detection to 70 μg/L by an injection volume of 160 μmL.

Full text of DOI:10.1021/ac034876a

Anal. Chem. 2004, 76, 137-143
Biosensor-Based Determination of Riboflavin in
Milk Samples
Isabelle Caelen,*,† Andras Kalman,† and Lennart Wahlstro1m‡
Nestle´ Research Center Lausanne, Nestec Ltd., Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland, and
Biacore AB, Rapsgatan 7, 754 50 Uppsala, Sweden
the binding with Rf binding protein (RBP) from egg white7-9 have
been investigated.
An assay for quantification of riboflavin (Rf) in milk-based
products has been developed using the principle of
surface plasmon resonance with on-chip measurement.
The quantification was done indirectly by measuring
excess of Rf binding protein (RBP ) that remains free after
complexation with Rf molecules originally present in the
sample solution. The chip was modified with covalently
immobilized Rf in order to bind the RBP in excess. A
chemical modification was performed to introduce a
reactive ester group at the N-3 position of the natural Rf
to bind amino groups present on the chip surface.
Calibration solutions were prepared by mixing a range of
Rf standard solutions with an optimized concentration of
RBP . The Rf content in the milk-based products was then
measured by comparison of the response against the
calibration. Results obtained were very close to those from
an official HP LC-fluorescence procedure. The limit of
quantification was determined to 2 3 4 µg/ L and the limit
of detection to 7 0 µg/ L by an injection volume of 1 6 0 µL.
Interest in developing analytical techniques based on selec-
tive biointeractions between biochemical entities increases day
after day. Development of a biosensor-based approach, using a
specific interaction between Rf and RBP10,11 in molecular ratio 1:1,
would appear to be competitive with classical approaches in terms
of speed of analysis and simplicity. In particular, the surface
plasmon resonance (SPR) technique, which detects changes in
mass onto the sensor chip surface provides quantitative, specific,
and real-time analysis and allows kinetic and affinity analysis.12
In the case of low molecular weight compounds, such as vitamins,
indirect assay is performed.13-17 The chip surface is first modified
with Rf derivative covalently immobilized on the surface. Next,
RBP is added in Rf containing samples where complexation of
the proteins occurs. Finally, the sample is injected onto the surface
and the free remaining RBP binds to the Rf derivative molecules
on the chip surface. A number of conditions need to be met to
obtain a successful SPR assay. First, the chemistry involved in
linking ligand molecules to surfaces should fully preserve their
biochemical properties. Second, immobilized ligands should be
easily accessible to ensure optimum detection and successive
quantification of target molecules. Third, the biochemical layer
must be stable, providing identical binding properties after many
steps of washing the surface and regeneration of the free ligand
molecules. In this work, the development of a new Rf assay is
described and evaluated for the quantification of Rf in milk-based
products, taking into account the three principles mentioned
above. This type of product was chosen as a test matrix since
more than a quarter of the average Rf intake is from milk and
milk products.
Riboflavin (Rf) is an essential vitamin in human nutrition
occurring in a wide variety of food products. In the body riboflavin
is transformed into two active coenzymes, flavin mononucleotide
(FMN) and flavin adenine dinucleotide (FAD). These two active
flavins are directly involved as coenzymes with oxidases and
dehydrogenases for the hydrolysis of fatty acids and the degrada-
tion of amino acids or pyruvic bases. Second, flavins can transfer
electrons or protons from a donor to an acceptor, important in
the Krebs cycle and the respiratory chain. Official methods for
quantification of Rf in food samples are based usually on HPLC
measurement and microbiological assays. Alternative methods
involve electrochemical or fluorescent characteristics of flavins
for standard solutions,1-3 biological fluids,4,5 and pharmaceutical
tablets6 but none for food matrixes. In addition to these ap-
proaches, methods based on biological properties of Rf through
(7) Becvar, J.; Palmer, G. J. Biol. Chem. 1 9 8 2 , 257, 5607-17.
(8) Lotter, S. E.; Miller, M. S.; Bruch, R. C.; White, H. B., III. Anal. Biochem.
1 9 8 2 , 125, 110-7.
(9) Ushijima, H.; Okamura, H.; Nishina, Y.; Shiga, K. J. Biochem. 1 9 8 9 , 105,
* To whom correspondence should be addressed. E-mail: isabelle.
caelen@rdls.nestle.com.
467-72.
(10) Ogasawara, F. K.; Wang, Y.; Bobbitt, D. R. Anal. Chem. 1 9 9 2 , 64, 1637-
42.
† Nestec Ltd.
‡ Biacore AB.
(11) Yao, T.; Rechnitz, G. A. Anal. Chem. 1 9 8 7 , 59, 2115-8.
(12) Lo¨ fas, S.; Johnsson, B. J. Chem. Soc., Chem. Commun. 1 9 9 0 , 1526-8.
(13) Caselunghe, M. B.; Lindeberg, J. Food Chem. 2 0 0 0 , 70, 523-32.
(14) Grace, T. A.; Stenberg, E. Cereal Foods World 2 0 0 2 , 47, 7.
(15) Haines, J.; Aulenta, F.; Patel, P. Leatherhead: LFRA 2 0 0 1 , 1-13.
(16) Indyk, H. E.; Evans, E. A.; Caselunghe, M. C. B.; Persson, B. S.; Finglas, P.
M.; Woollard, D. C.; Filonzi, E. L. J. AOAC Int. 2 0 0 0 , 83, 1141-8.
(17) Indyk, H. E.; Persson, B. S.; Malin, C. B.; Moberg, A.; Filonzi, E. L.; Woollard,
D. C. J. AOAC Int. 2 0 0 1 , 85, 73-81.
(1) Nishikimi, M.; Kyogoku, Y J. Biochem. 1 9 7 3 , 73, 1233-42.
(2) Sawamoto, H. J. Electroanal. Chem. 1 9 8 5 , 186, 257-65.
(3) Wang, J.; Luo, D.-B.; Farias, P. A. M.; Mahmoud, J. S. Anal. Chem. 1 9 8 5 ,
57, 158-62.
(4) Kozik, A. Analyst 1 9 9 6 , 121, 333-37.
(5) Kodentsova, V. M.; Vrzhesinskaya, O. A.; Spirichev, V. B. Ann. Nutr. Metab.
1 9 9 5 , 39, 355-60.
(6) Zhang, C.; Qi, H. Anal. Sci. 2 0 0 2 , 18, 819-22.
10.1021/ac034876a CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/14/2003
Analytical Chemistry, Vol. 76, No. 1, January 1, 2004 137

MATERIALS AND METHODS
to solvent signal. One-dimensional 1H NMR, 13C NMR, and
distortionless enhancement by polarization transfer (DEPT 135),
were acquired using standard conditions.22 The spectra were
interpreted using the MestRe-C 2.3 software.
Materials. Riboflavin was purchased from Calbiochem (Darm-
stadt, Germany); riboflavin binding protein and retinol binding
protein (ABP) were from Fluka (Buchs, Switzerland). All the
organic solutions were purchased from Merck (Buchs, Switzer-
land) or Fluka.
For the acetylation: 1H NMR (DMSO-d8) δ 1.93-2.42 (6 s, 18H,
4CH3CO, 2CH3), 4.11-4.17 (m, 1H, CH2), 4.27-4.30 (m, 1H, CH2),
4.52-4.55 (m, 1H, CH2), 4.71-4.75 (m, 1H, CH2), 5.13-5.38 (m,
3H, 3 CH), 7.60 (s, 1H, aromatic), 7.74 (s, 1H, aromatic), 11.34
(s, 1H, NH); 13C NMR (DMSO-d8) δ 19.6-21.4 (6CH3, 4CH3CO,
2CH3), 44.5 (NCH2), 62.3 (1CH2, OCH2), 69.6 (CH, OCH), 70.5
(CH, OCH), 73.4 (CH, OCH), 117.1 (CH, aromatic), 131.9 (CH,
aromatic), 134.3-137.5 (4Cq, aromatic), 147.2 (1Cq, CdN), 151.3
(1Cq, CdN), 156.2 (1Cq, NCdO), 160.6 (1Cq, NCdO), 170.3-
171.0 (4 Cq, CdO).
Methods. Riboflavin Derivative Synthesis. Protection of
Hydroxyl Groups. A 5.0-g sample of riboflavin was added to a 400-
mL beaker containing 200 mL of a 1:1 (v/ v) mixture of glacial
acetic acid and acetic anhydride. After the dropwise addition of 1
mL of 70% perchloric acid, the mixture was stirred for 30 min at
40 °C.10 The mixture was cooled in an ice bath and diluted with
an equal volume of cold water, and the solution was then extracted
twice with 400 mL of chloroform. The combined chloroform
extracts were washed twice with 200 mL of deionized water and
the organic phase dried over anhydrous sodium sulfate.18 After
filtration, the solvent was removed under reduced pressure at 50
°C to a yield of 95% with a melting point of 219-220 °C.
Derivatization at the N-3 Position. A 1 mol equiv of 2′,3′,4′,5′-
tetra-O-acetylriboflavin was reacted with bromoacetic acid ethyl
ester (5 mol equiv) with stirring for 20 h at room temperature in
anhydrous N,N-dimethylformamide containing anhydrous potas-
sium carbonate (5 mol equiv). Solvent was removed by evaporation
under reduced pressure with warming, and the residue was
redissolved in an equal volume of methylene chloride and washed
twice with an equivalent volume of 1 N acetic acid and then twice
with water.19 The organic phase was dried over anhydrous sodium
sulfate, and the methylene chloride was removed overnight under
reduced pressure at 36 °C to a yield of 73%.
1
For the derivatization on N-3: H NMR (DMSO-d8) δ 1.1 (q,
3H, CH3), 1.93-2.46 (6 s, 18 H, 4CH3CO, 2CH3), 3.26 (s, 2H, CH2),
4.03-4.30 (m, 2H, CH2), 4.55-4.75 (m, 2H, CH2), 5.13-5.38 (m,
3H, 3 CH), 7.72 (s, 1H, aromatic), 7.78 (s, 1H, aromatic); 13C NMR
(DMSO-d8) δ 14.2 (CH3, 19.5-21.6), (6 CH3, 4CH3CO, 2CH3), 43.2
(NCH2), 44.9 (NCH2), 61.4 (1CH2, OCH2), 63.3 (1CH2, OCH2), 69.5
(CH, OCH), 70.5 (CH, OCH), 73.4 (CH, OCH), 117.1 (CH,
aromatic), 131.9 (CH, aromatic), 135.1-137.5 (4Cq, aromatic), 47.2
(1Cq, CdN), 151.3 (1Cq, CdN), 154.8 (1Cq, NCdO), 159.9 (1Cq,
NCdO), 168.1 (Cq, Cdo), 170.3-171.0 (4Cq, CdO).
For the deprotection: 1H NMR (D2O) δ 2.21 (s, 3H, CH3), 2.39
(s, 3H, CH3), 3.66-3.74 (m, 1H, CH2), 3.82-3.95 (m, 1H, CH2),
4.11-4.37 (m, 4H, 2 CH2), 4.46-4.63 (m, 3H, CH), 7.38 (s, 1H,
aromatic), 7.65 (s, 1H, aromatic); 13C NMR (D2O) δ 19.6 (1CH3),
21.4 (1CH3), 43.2 (NCH2), 44.2 (NCH2), 60.1 (1CH2, OCH2), 69.6
(CH, OCH), 72.8 (CH, OCH), 73.7 (CH, OCH), 125.2 (CH,
aromatic), 139.7 (CH, aromatic), 130.7-135.0 (4 Cq, aromatic),
148.9 (1Cq, CdN), 151.3 (1Cq, CdN), 156.6 (1Cq, NCdO), 160.8
(1Cq, NCdO), 174.8 (1Cq, CdO).
Deprotection of the Hydroxyl Groups. The residue was suspended
in 2 N HCl (equal volume of previous step), refluxed for 2 h
and evaporated to dryness.20
Formation of Activated Ester. A 0.50-mmol aliquot of N,N′-
dicyclohexylcarbidiimide (DCC) was added to a solution of
3-carboxymethylriboflavin (0.55 mmol) and N-hydroxysulfosuc-
cinimide sodium salt (sulfo-NHS; 0.50 mmol) in 15 mL of
anhydrous DMF at 0 °C, 48 h in the dark (to prevent photodeg-
radation of the N-10 side chain of Rf) at 4 °C under stirring.
Dicyclohexylurea formed during the reaction was removed by
filtration.
The activated product was isolated by evaporating of DMF at
reduced pressure and by washing the resulting residue with
ethanol. Any remaining DMF, DCC, or sulfo-NHS was removed
by ethanol, a solvent in which the activated ester product is not
soluble. The activated ester (powder form) prepared in this
manner was stable for at least 6 months when stored below
0 °C.21
NMR Analysis. Each step of organic synthesis was followed
by NMR analysis. The samples for NMR spectroscopy were
prepared in Wilmad 528-PP 5-mm Pyrex NMR tubes, using heavy
water or DMSO as solvent (0.7 mL). The NMR spectra were
acquired on a Bruker AM-360 spectrometer, equipped with a
quadrinuclear 5-mm probe head, at 360.13 MHz for 1H and at 90.03
MHz for 13C spectra. All shifts are cited in ppm measured relative
Fluorescence Measurements. A stock solution of 1 M
phosphate buffer (pH 7.4) was prepared monthly, and dilutions
to 0.1 and 0.01 M were used. A 2.0-mg sample of Rf derivative
(13.7 µM) was reacted under agitation for 1 h at room temperature
in 230.5 mL of 0.1 M phosphate buffer with 3 mol equiv
ethanolamine. This solution of blocked Rf was diluted 4 times
(concentration of 3.42 µM) in 0.1 M phosphate buffer. A 95-µL
sample of Rf and 5 µL of different concentrations of RBP or ABP
were introduced in the wells of a microtiter plate. The analysis
was performed with a SpectraFluor Plus (Tecan, Hombrechtikon,
Switzerland) for a fluorescent light setup at 360 nm and an
emission light read at 535 nm.
P reparation of the Chip Surface and SP R Analysis. Sur-
face immobilization and analysis were performed simultaneously
using a Biacore Q system (Biacore AB, Uppsala, Sweden) using
a carboxymethylated dextran sensor chip (CM5, Biacore AB).
HBS-EP (Biacore AB) buffer was used as running buffer for all
immobilizations. The measurements are real-time analysis giving
curves named “sensorgrams”. These sensorgrams are a plot of
response (arbitrary units) as a function of time (seconds). One
arbitrary unit (au) increase corresponds to 1 pg/ mm2 increase of
surface density.
(18) McCormick, D. B. J. Heterocycl. Chem. 1 9 7 0 , 7, 447-50.
(19) Wu, F. Y-H.; MacKenzie, R. E.; McCormick, D. B. Biochemistry 1 9 7 0 , 9,
2219-24.
(20) Merril, A. M.; McCormick, D. B. Methods. Enzymol. 1 9 8 0 , 66, 338-44.
(21) Cha, G. S.; Meyerhoff, M. E. Anal. Biochem. 1 9 8 8 , 168, 216-27.
(22) Lin, J.; Welti, D. H.; Arce Vera, F.; Fay, L. B.; Blank, I. J. Agric. Food Chem.
1 9 9 9 , 47, 2813-21.
138 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

Derivatization of Dextran End Groups. The gold surface of the
CM5 chip with carboxyl-end molecules of dextran was washed
with deionized water. Solutions of N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide/ N-hydroxysuccinimide ester (EDC/ NHS) (0.4
M/ 0.1 M in water) from the amine coupling kit (Biacore AB) were
injected on the surface (flow rate 10 µL/ min, contact time 7 min).
Dihydrochloride ethylenediamine solution (0.1 M in 50 mM borate
solution) was injected on the surface (flow rate 10 µL/ min, contact
time 7 min). Remaining ester was deactivated with 1 M ethanol-
amine in water (pH 8.5) (flow rate 19 µL/ min, contact time 3
min). Washing of the surface was done with 1 M NaCl (flow
rate 20 µL/ min, contact time 4 min) and with water (flow rate
20 µL/ min, contact time 4 min).
Immobilization of Riboflavin Derivative. Buffer (0.1 M bis-tris
propane in 0.01 M phosphate buffer, pH 6.5) was prepared for
the immobilization of riboflavin. A 5 mM concentration of Rf
derivative in cold immobilization buffer was injected on the surface
(flow rate 10 µL/ min) for 7 min. The surface was washed with 1
M ethanolamine hydrochloride (pH 8.5) (Biacore AB) at a flow
rate of 19 µL/ min for 7 min to remove any unbound ester of Rf
derivative.
RBP Binding. Injection of sample solutions (pure RBP solutions
or mixed solutions containing riboflavin and a known amount of
RBP) was done for 8 min at a flow rate of 20 µL/ min. The solution
was mixed automatically in the Biacore Q instrument, using 75%
of RBP solution and 25% of sample solutions. Solutions of RBP
were prepared daily in 0.1 M phosphate buffer containing 0.6 M
NaCl and 0.1% surfactant P20 (Biacore AB). For the surface
regeneration, 0.033% SDS23 (pH 5) was used for 50 s at a flow
rate of 40 µL/ min.
Sample Preparation. The sample preparation procedure fol-
lowed the official French method that describes the determination
of vitamins B1 and B2 by reversed-phase high-pressure liquid
chromatography (RP-HPLC). This method applies an enzymatic
hydrolysis followed by RP-HPLC with fluorescence detection, and
it is validated for fortified food products such as infant formulas,
infant cereals, milk-based dietetic products, breakfast cereals, and
fortified drinks. Based on the French HPLC method, a CEN
method is now under approval.24
manner to the complexation with RBP molecules,7 and hence, they
need to be preserved. First, some hydrophobic interactions occur
via the two methyl groups at the C-7 and C-8 positions of the Rf
molecule. This dimethylbenzenoid portion of the ring is involved
in primary interactions of binding and is relatively buried in the
interacting RBP, and both methyl groups seem to facilitate a fit
with the binding site that maintains the proper position of the
flavin ring system. Second, steric limitation is imposed at C-8, N-10,
especially C-1′, 2′-hydroxyl group of the ribytil chain, and 4-car-
bonyl positions.26 Last, the hydroxyl groups of the N-10 side chain
contribute in a stereoselective manner by formation of hydrogen
bonds. However, substituents at C-2 and N-3 do not compromise
binding affinity between the partners, and thus, derivatization can
occur at these positions.27 In the case of RBP, a tryptophanyl
residue is essential for the binding with Rf, and in addition, a
tyrosine group is probably involved in a stack interaction with
flavin molecules.28,29 As a consequence, a functional reactive group
at the N-3 position was chosen that could bind surface molecules.
Briefly, acetylation of hydroxyl groups was performed (compound
2 ) followed by nucleophilic substitution of the nitrogen atom on
a bromoacetic acid ethyl ester (compound 3 ). The carboxylic acid
(compound 4 ) obtained by hydrolysis of this ester is then
converted back into a highly reactive ester (compound 5 ). Each
step of the synthesis was confirmed by NMR analysis. This
derivative can subsequently react by forming an amide linkage
with free amino groups on the surface.
Natural fluorescence of the Rf molecule is quenched by the
tryptophanyl residue of RBP during complexation.30 Hence,
bioreaction between Rf derivative and RBP can be evaluated by
measuring quenching of Rf fluorescence when mixed in solution.
Reactive ester groups on the derivative molecules that bind amino
groups were inactivated with ethanolamine prior to the addition
of RBP to prevent any covalent binding between the two biomol-
ecules. Covalent binding itself could be responsible for a decrease
of emitted fluorescence, falsifying the measurement of quenching
due to the biointeractions. Fluorescence signals of the native Rf
or the Rf derivative were followed by homogeneous assays as
shown in Figure 2. Solutions of equal concentrations of Rf and its
derivative were introduced separately into the wells of a microtiter
plate, with increasing amount of RBP molecules. An increase in
the concentration of RBP leads to an increase of fluorescence
quenching for both Rf and Rf derivative. Negative tests demon-
strate that binding does not occur between the two kinds of Rf
molecules and ABP. It was observed that progressively increasing
the concentration of ABP in the wells does not lower the level of
fluorescence. As a consequence, quenching of fluorescence
observed with RBP is certainly due to the biointeraction between
RBP and riboflavins. The curves corresponding to Rf and Rf
derivative are superposable up to a concentration of RBP of ∼1.5
µM. The fluorescence quenching of Rf and Rf derivative differs
at higher concentration of RBP, which could be due to impurities
of the solution of Rf derivative.
Our sample preparation procedure was made similarly accord-
ing to the treatment with Taka-Diastase enzyme (Fluka) at
45-50 °C to break up compounds with high molecular masses.
This was followed by acidic hydrolysis for 30 min in boiling water
bath with 1 M HCl solution. After cooling the solution to room
temperature, it was filtered through a 0.22-µm filter so that the
solution was ready for the assay.
RESULTS AND DISCUSSION
Synthesis of Riboflavin Derivative and Test for Chemical
Functionality. Natural riboflavin does not contain chemical
groups that are able to bind to the chip surface, and therefore,
organic synthesis is performed to introduce reactive ester groups
at the N-3 position of the molecule.25 A summary of the four steps
synthesis of the Rf derivative is shown in Figure 1. Several groups
on the Rf nucleus (compound 1 ) contribute in a nonselective
Immobilization Chemistry of Riboflavin Derivative. This
step illustrates the covalent immobilization of riboflavin derivative
(26) Mifflin, T. E.; Langerman, N. Arch. Biochem. Biophys. 1 9 8 3 , 224, 319-25.
(27) Choi, J. D.; McCormick, D. B. Arch. Biochem. Biophys. 1 9 8 0 , 204, 41-51.
(28) Monaco, H. L. EMBO J. 1 9 9 7 , 16, 1475-83.
(23) Murthy, U. S.; Podder, S. K.; Adiga, P. R. Biochim. Biophys. Acta 1 9 7 6 ,
434, 69-81.
(24) CEN/ TC 275 Work programme, pr EN 14 152 (under approval).
(29) Blankenhorn, G. Eur. J. Biochem. 1 9 7 8 , 82, 155-60.
(25) Merril, A. M.; McCormick, D. B. Anal. Biochem. 1 9 7 8 , 89, 87-102.
(30) Guevara, I.; Zak, Z. J. Protein Chem. 1 9 9 3 , 12, 179-85.
Analytical Chemistry, Vol. 76, No. 1, January 1, 2004 139

Figure 1. Synthesis of riboflavin derivative.
enhanced stability, increasing the time for primary amino groups
to react with the ester. In the third step, Rf derivatives were
dissolved in cold bis-tris propane buffer to reduce the rate of
hydrolysis of the activated ester prior to the reaction with amino
groups on the surface, and the solution is injected onto the amine
surface. The immobilization procedure was followed after each
step by SPR measurement, Figure 3B, showing an increase of
the response (value related to the refractive index) from step 1
to step 3. This trend is explained by the variation of surface-
associated mass, due to successive immobilizations of EDA and
Rf derivative molecules: surface density is around 190 pg/ mm2
for the amino surface and 350 pg/ mm2 for the surface-immobilized
Rf. Finally, the surface was intensively washed while keeping the
same response, meaning that Rf derivatives were covalently bound
on the surface.
Figure 2. Fluorescence quenching of (a) Rf and (b) Rf derivative
in homogeneous assay, validating the biological properties of the
molecules. Percentage of fluorescence quenching for riboflavin and
its derivative is shown as a function of RBP concentration in solution.
Molecular ratio between Rf or Rf derivative and RBP is indicated on
the top of the curves. The lower curves represent reaction between
ABP with (a′) Rf and (b′) Rf derivative.
Detection and Binding of RBP . Injecting different concentra-
tions of the RBP onto the chip surface, Figure 4, was performed
to test the ability of the immobilized Rf on the surface to be
complexed by RBP. The response is followed in situ by SPR
measurement as a function of time after injection of RBP solution,
Figure 4A. Surface-associated mass increased with increasing
concentration of RBP, for a range of concentrations between 0.1
and 10 µg/ mL. Figure 4B shows the final response level, after
rinsing of the surface, as a function of RBP concentration. The
average values from three different experiments exhibit an
exponential relationship when plotted on a logarithmic scale, up
to a concentration of RBP of 10 µg/ mL. The inset curve presents
onto the chip surface on which a dextran polymer bearing carboxyl
groups is bound, Figure 3A. Injection of a solution of ethylene-
diamine (EDA) was performed to transform carboxyl end groups
into amino groups via the formation of an amide linkage between
the molecules. An excess of EDA prevented the molecules from
reacting at both amino ends, leading to an unreactive surface. An
increased coupling yield between EDA and carboxyl groups can
be obtained by using NHS in conjunction with carbodiimide:
O-acylisourea intermediate formed between EDC and carboxyl
groups reacts with NHS to produce an active ester with an
140 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

Figure 3. Riboflavin derivative molecules covalently bound on the chip surface. (A) Carboxyl-end surface is transformed into amino-end
surface after reaction with ethylenediamine. Riboflavin derivative can subsequently react by forming an amide linkage with primary amino groups
on the surface. (B) Response associated with the measurement of refractive index at the surface is followed after each step of immobilization,
demonstrating an increase of the mass present at the surface from step 1 to step 3.
Figure 5. Response obtained after complexation of RBP (open
symbols) is compared to response obtained by direct assay (filled
symbols) for two different concentrations of RBP: (A) 500 ng/mL
(4, 2) and (B) 10 µg/mL (O, b).
conditions completely releases the RBP molecules from the chip
surface, and successive cycles of binding and regeneration may
be performed.
Figure 4. Direct binding assay of RBP to immobilized Rf derivative
Assay for Quantification of Riboflavin in Food Samples.
performed using different concentrations of binding protein. (A) The
The next set of experiments characterizes the complexation
response is followed in situ after injection of RBP with concentrations
reaction of the RBP when mixed with samples containing riboflavin
of (a) 0.1, (b) 0.2, (c) 0.4, (d) 0.8, (e) 1, (f) 2.5, (g) 5, and (h) 10
molecules. A comparison was made between injections of RBP
molecules directly onto the surface with residual RBP molecules
after complexation with Rf molecules, Figure 5. Concentrations
of free RBP at 500 ng/ mL and 10 µg/ mL were chosen, to compare
values at low and high response according to the results from
Figure 4B. In the case of complexation reaction, an initial
concentration of Rf standard solution at 500 ng/ mL was mixed in
appropriate ratio with a total concentration of the solution of RBP
at 20 and 10.5 µg/ mL. As a result, the responses obtained for the
two experiments are very similar for both low and high free RBP
concentrations. The final response value after 8 min for the
µg/mL. (B) Responses obtained after intensive washing of the surface
are plotted as a function of RBP concentration. The inset drawing
corresponds to linear range of the response up to a concentration of
1000 ng/mL.
linear response as function of RBP concentration up to a
concentration level of 1 µg/ mL. These results demonstrate, first,
that the RBP binds efficiently to the surface-immobilized Rf and,
second, that the quantity of Rf derivatives on the surface is
adequate to discriminate between different concentrations of RBP
molecules from the solution. A regeneration step using acidic
Analytical Chemistry, Vol. 76, No. 1, January 1, 2004 141

Figure 6. Complexation assay for RBP to quantify Rf in food samples. (A) Calibration curve is obtained for a panel of Rf concentrations
obtained by applying standard extraction procedures (HPLC-based) from food matrixes. (B) Standard solutions of Rf were injected at low
concentration range from 3 to 400 ng/mL on the surface to determine limits of detection (LOD) and quantification (LOQ).
complexation assay was 10% less than the response obtained with
Table 1. Comparison of the Results of SPR
RBP directly injected on the surface. First, this test shows that
RBP was efficiently complexed with Rf molecules and, second,
that the binding of RBP on the surface is not altered in the
presence of these bound molecules in the solution.
Measurement with HPLC Reference Values
riboflavin concn in
mg/ 100 g measured by
SPR
HPLC
Figure 6A shows a plot of response as a function of Rf
concentration between 200 and 1000 ng/ mL, with a total concen-
tration of RBP at 19.3 µg/ mL. This range corresponds to free
concentrations of RBP from 15.3 to 0 µg/ mL. Calibration curves
have a sigmoidal shape, and consequently, quantification over a
quite large concentration range is possible. The coefficient of
variation between two different calibration solutions at the same
concentration is below 5%. To minimize the possible matrix effect
of the sample on the RBP binding on the chip surface, we chose
to dilute three times the sample solution in the RBP solution prior
the injection. The experimental responses associated with this
range of concentration of Rf are lower than those calculated, based
on the results of Figure 4B, except for the value at 1000 ng/ mL
which should be ∼0. Indeed, binding of RBP on the surface occurs
for a Rf concentration of 1000 ng/ mL (response ∼100), showing
that complexation of RBP is not fully completed. Ideally, the
response obtained for the Rf-containing samples should be in
the linear region of the curve in order to reach a maximal
sensitivity and good separation of values for similar concentrations
of Rf. In Figure 6B, limits of detection and quantification were
determined. Using the same experimental conditions, solutions
with different low concentrations of Rf, from 3 to 400 ng/ mL,
were injected on the surface with a concentration of RBP of 19.3
ng/ mL and an injection volume of 160 µL 1 day after the pre-
vious measurement. The values obtained for 200 and 400 ng/ mL
RBP were higher (∼100 au) than those obtained previously
because the initial baseline measured on the chip had an increase
of ∼100 au. The matrix on the sensor chip consists of linear
dextran, which is swollen in aqueous media, providing an
extensively solvated hydrogel. When stocked overnight, the level
of hydration of the chip surface changes, and hence the initial
baseline can vary, affecting the absolute measurements but not
the relative ones. As a consequence, it is advised to run the sam-
ple measurements the same day as the calibration curve. Injec-
tion of five blank samples was performed, and the corresponding
value of standard deviation was 5%. Under those experimental
conditions, the limit of detection (LOD) was 70 ng/ mL and
the limit of quantification (LOQ) was 234 ng/ mL for Rf solu-
tion. This calibration was applied to the determination of Rf in
milk-based product a
milk-based product b
1.67 ( 0.09
1.42 ( 0.02
1.69 ( 0.17
1.38 ( 0.14
two milk samples (a, b), and the results were in good agreement
with those given by official method as indicated in Table 1. Rf
was extracted from the milk-based products according to the
official procedure described in Materials and Methods. Sample
extracts were diluted to obtain a Rf concentration between 500
and 1000 ng/ mL. The samples gave 238 and 365 au in response,
respectively (duplicate analysis for each sample), in the region of
the calibration curve where it is possible to distinguish close values
(see Figure 6A).
CONCLUSIONS
This work describes the successful development of a SPR assay
to quantify riboflavin in milk samples based on complexation of
Rf binding protein added to the sample solution. The excess of
RBP binds to the ligand molecule, a derivative of Rf that was
synthesized and subsequently immobilized on the chip surface.
Even though the initial results obtained by SPR are in good
agreement with HPLC data, further work was required to optimize
the concentration of added RBP in conjunction with a suitable
range for Rf calibration in order to decrease the LOD and the
LOQ. Another route for the synthesis of Rf derivative was used
to increase the purity of the compound and hence the binding
capacity of the surface. Under those conditions, the LOD de-
creased down to 17.1 ng/ mL and LOQ down to 42.9 ng/ mL. The
repeatability of the assay is under 3% and the reproductibility under
4.5%.31 Once the derivative is immobilized on the surface, at least
400 assays can be performed on the same chip, which has a
lifetime of 3 months when stored under 4 °C.
Conventional assays include microbiological assays (MBA) and
HPLC. MBA is limited by poor reproductibility, long sample
preparation, and analysis time of 2-3 days. The HPLC method
requires lengthly sample preparation and a sample turnover
(31) Kalman, A.; O’Kane, A.; Caelen, I.; Trisconi, M.-J.; Wahlstro¨ m; L., submitted
to New-Food.
142 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

limited to 24 samples per 24 h. Biosensor measurements based
on SPR principles present a number of advantages: automatic
mixing and injection of the solutions make the system friendly to
use, and the final concentrations of Rf in the samples are directly
calculated by the software, giving fast and reliable measurements.
Downscale measurement on chip surfaces economize reagents,
shorten analysis time, and offer multidetermination of analytes in
a single experiment. Using the optimized procedure described
above, 20 samples can be fully analyzed in less than 6 h and up
to 88 samples can be measured in a single run. Biosensor-based
assays propose an alternative method to traditional analytical
techniques.
ACKNOWLEDGMENT
We thank Dr. J. Svorc for useful discussions and interest in
the work and Dr. O. Heudi for his contribution to the writing of
the article. We express our thanks to Mrs. M.-J. Trisconi for her
help in the sample preparation. We warmly thank Dr. F. Robert
and Mr. W. Matthey-Doret for their precious advise in the organic
synthesis. We are grateful to Dr. J.C. Blake for carefully reading
the manuscript and for his continuous support.
Received for review July 30, 2003. Accepted October 14,
2003.
AC034876A
Analytical Chemistry, Vol. 76, No. 1, January 1, 2004 143