Anal. Chem.
XXXX, xxx, 000–000
Folic Acid-Anchored PEGgylated Phospholipid
Bioconjugate and Its Application in a Liposomal
Immunodiagnostic Assay for Folic Acid
Ja-an Annie Ho,*,† Chi-Hsiang Hung,† Li-Chen Wu,‡ and Ming-Yuan Liao§
BioAnalytical Lab, Department of Chemistry, National Tsing Hua University, Hsin-chu, 300 Taiwan, Biochemistry,
Department of Applied Chemistry, National Chi Nan University, Puli, 545 Taiwan, and Department of Chemistry,
National Chung Hsing University, Taichung, 402 Taiwan
A folic acid-anchored, poly(ethylene glycol)-linked (PEG-
gylated) phospholipid and an immunoaffinity chromato-
graphic column were prepared and employed to develop
a liposomal immunodiagnostic assay for the direct deter-
mination of folic acid (FA) in this study. Distearoylphos-
phatidylethanolamine-poly(ethylene glycol)2000-folic acid
(DSPE-PEG2000-FA) was synthesized through carbo-
diimide-mediated coupling of FA and DSPE-PEG2000-
amine and characterized using thin layer chromatog-
raphy, 1H nuclear magnetic resonance spectroscopy,
and electrospray ionization-mass spectrometry. Lipo-
somal biolabels were constructed using the synthesized
DSPE-PEG2000-FA in conjunction with other phospho-
lipids. A stationary phase having affinity for FA was
prepared by covalently linking purified anti-FA mono-
clonal antibodies onto N-hydroxysuccinimide-activated
Sepharose beads, which were subsequently packed
into a 1.9 cm diameter polypropylene column. The
calibration curve for FA had a linear range from 10-8
to 10-4 M. The limit of detection was 6.8 ng (equivalent
to 500 µL of 3.1 × 10-8 M FA). The elution buffer
(35% methanol in Tris buffered saline containing 0.1%
Tween 20) also served as the regeneration buffer,
which allowed the same column to be used for up to
50 times without any observable loss of reactivity. The
immunoaffinity chromatographic column was reusable
and capable of concentrating analytes from sample
solution; in conjunction with folic acid-sensitized lipo-
somal biolabels, however, they hold great potential as
sensitive immunoaffinity assays for the determination
for FA. To confirm the feasibility of using this system
in the analysis of real samples, the folic acid contents
of three over-the-counter vitamin supplements were
tested. The recoveries of folic acid of 90-112% for
these three samples were obtained, suggesting co-
ntents that were consistent with the information ob-
tained from their nutritional facts panels.
* Corresponding author. Fax: +886-3-571-1082. E-mail: jaho@mx.nthu.edu.tw.
†
National Tsing Hua University.
‡
National Chi Nan University.
§
National Chung Hsing University.
10.1021/ac900402v CCC: $40.75  XXXX American Chemical Society
Folic acid (FA) was originally extracted from yeast by Willis1
in 1931; it was initially called vitamin M or vitamin Bc. Technically,
the name “folic acid” refers to the manmade type of B vitamin
that is used in vitamin supplements or added to certain foods (so-
called enriched or fortified foods) and “folate” refers to the natural
type of B vitamin, which is already present in some foods.
Nowadays, however, folate is a generic term that refers to all
derivatives of folic acid.
Biologically, FA is essential for transferring one-carbon units
involved in phospholipid, DNA, protein, and neurotransmitter
syntheses.2 Folate is an essential dietary component for the
formation of red and white blood cells and the epithelial cells in
the digestive tract.3 At present, adequate folate nutriture is
encouraged because of the possible correlation between folate
intake and the occurrence of pregnancy neural tube defects and
occlusive vascular disease, where an increased concentration of
homocysteine in the blood4,5 has been implicated as a risk factor.6,7
Therefore, sufficient folate nutriture is believed to reduce the
incidence of cardiovascular disease by lowering homocysteine
concentrations in plasma and serum. Furthermore, Selhub and
Rosenberg (1996)2 observed a close relationship between folate
and choline in the reactions of the methyl cycle. Moreover, folate
intake appears to be inversely related to the risk of colorectal
cancer.8 In light of these health benefits, in January 1998, the U.S.
Food and Drug Administration mandated9 the fortification of
cereal grain products with FA at a level of 140 mg per 100 g, which
is expected to yield a reduction in the incidence of folate-associated
diseases in the U.S.10 The average folate intake of adult men and
(1) Willis, S. L. Br. Med. J. 1931, 1, 1059–1064.
(2) Selhub, J.; Rosenberg, I. H. Folic Acid. In Present Knowledge in Nutrition,
7th ed.; Ziegler, E. E., Filer L. J., Jr., Eds.; International Life Sciences
Institute Press: Washington, DC, 1996; pp 206-219.
(3) Gregory, J. F.; Ristow, K. A.; Sartain, D. B.; Damron, B. L. J. Agric. Food
Chem. 1984, 32, 1337–1342.
(4) Wald, N. Lancet 1991, 338, 131–137.
(5) Boushey, C. J.; Beresford, S. A. A.; Omenn, G. S.; Motulsky, A. G. JAMA
1995, 274, 1049–1057.
(6) Hankey, G. J.; Eikelboom, J. W. Lancet 1999, 354, 407–413.
(7) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu. Rev. Med. 1998,
49, 31–62.
(8) Johnson, I. T.; Lund, E. K. Aliment. Pharmacol. Ther. 2007, 26, 161–181.
(9) Food and Drug Administration (FDA). Food Standards: Amendment of
Standards of Identity for Enriched Grain Products to Require Addition of
Folic Acid; Final Rule (21 CFR Parts 136, 137, and 139). Fed. Regist. 1996,
61, 8781-8797.
(10) Tucker, K. L.; Mahnken, B.; Wilson, P. W. F.; Jacques, P.; Selhub, J. JAMA
1996, 276, 1879–1885.
Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX A
women in various European countries ranges from 150 to 400
µg/day.11,12
The existence of folate in many different chemical forms (with
various stabilities) in foods makes it considerably difficult to
characterize the vitamin and establish accurate data regarding its
levels. Over the past four decades, many techniques have been
employed in the analysis of folate, including microbiological,13-16
radiobinding,17 radiometric,18 electrochemical,19-21 and high-
performance liquid chromatography-based13,21-25 assays, immuno-
assays,26,27 and optical biosensing devices based on surface
plasmon resonance (SPR).28-30 Microbiological assays (MBAs)
are used most widely; they are considered among the most
versatile approaches to determining food folate levels. The
principle of MBAs is based on the quantitative relationship
between folate content and the growth of certain microorganisms,
including Lactobacillus casei (L. casei, ATCC 7469), Streptococcus
faecium (ATCC 8043), and Leuconostoc citrovorum (ATCC
8081).13,14,16 Among these microorganisms, L. casei is used most
widely for determining food folate levels because it responds
almost equally to the widest variety of folate derivatives. Despite
their popular use, MBAs are particularly laborious and time-
consuming, making it difficult to be established as reliable routine
laboratory methods. Although liquid chromatography/mass spec-
trometry-based analysis has been developed in recent years to
determine food folate levels, it remains expensive and not
accessible to all research laboratories. The relatively low accura-
cies of the established methods and their complicated sample
preparation requirements, often including extraction, deconjuga-
tion, and purification, mean that none of them have attained official
status as a reference method for the measurement of natural folate
in food.31-33
The past decade has witnessed rapid growth in the develop-
ment of immunoassays for the screening of biologically and
environmentally important target molecules. Because of their high
(11) de Bree, A.; van Dusseldorp, M.; Brouwer, I. A.; van het Hof, K. H.; Steegers-
Theunissen, R. P. Eur. J. Clin. Nutr. 1997, 51, 643–660.
(12) Hawkes, J. G.; Villota, R. Crit. Rev. Food Sci. Nutr. 1989, 28, 439–538.
(13) Tamura, T. J. Nutr. Biochem. 1998, 9, 285–293.
(14) Horne, D. W. Methods Enzymol. 1997, 281, 38–43.
(15) Finglas, P. M.; Faure, U.; Southgate, D. A. T. Food Chem. 1993, 46, 199–
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(16) Horne, D. W.; Patterson, D. Clin. Chem. 1988, 34, 2357–2359.
(17) Desouza, S.; Eitenmiller, R. J. Micronutr. Anal. 1990, 7, 37–57.
(18) Chen, M. F.; Hill, J. W.; Mcintyre, P. A. J. Nutr. 1983, 113, 2192–2196.
(19) Shin, H. C.; Shimoda, M.; Kokue, E. J. Chromatogr., B: Biomed. Sci Appl.
1994, 661, 237–244.
(20) White, D. R.; Lee, H. S.; Kruger, R. E. J. Agric. Food Chem. 1991, 39,
714–717.
(21) Bagley, P. J.; Selhub, J. Clin. Chem. 2000, 46, 404–411.
(22) Osseyi, E. S.; Wehling, R. L.; Albrecht, J. A. J. Chromatogr., A 1998, 826,
235–240.
(23) Vahteristo, L. T.; Ollilainen, V.; Koivistoinen, P. E.; Varo, P. J. Agric. Food
Chem. 1996, 44, 477–482.
(24) Gounelle, J. C.; Ladjimi, H.; Prognon, P. Anal. Biochem. 1989, 176, 406–
411.
(25) Konings, E. J. M. J. AOAC Int. 1999, 82, 119–127.
(26) Das Sarma, J.; Duttagupta, C.; Ali, E.; Dhar, T. K. J. Immunol. Methods 1995,
184 (1), 1–6.
(27) Lermo, A.; Fabiano, S.; Hernandez, S.; Galve, R.; Marco, M. P.; Alegret, S.;
Pividori, M. I. Biosens. Bioelectron. 2009, 24 (7), 2057–2063.
(28) Caselunghe, M. C. B.; Lindeberg, J. Food Chem. 2000, 70 (4), 523–532.
(29) Gao, Y.; Guo, F.; Gokavi, S.; Chow, A.; Sheng, Q.; Guo, M. Food Chem.
2008, 110 (3), 769–776.
(30) 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. 2000, 83 (5), 1141–
1148.
B Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX
sensitivity, rapidity, and simplicity of operation, immunoassays are
among the most powerful tools for selectively detecting various
physiological, biological, and environmental substances. Because
the demand for food folate determination is increasing gradually,
there is an ongoing need for simpler, faster, less expensive, and
more reliable and user-friendly analytical tools. In this paper, we
present a simple method for preparing FA-anchored, poly(ethylene
glycol)-linked (PEGgylated) phospholipid bioconjugates, which
were subsequently incorporated into the formation of FA-anchored
liposomes. We have also developed a simple yet novel alternative
method for the detection of FA, one that combines an immunoaf-
finity chromatographic (IAC) assay with FA-anchored liposomal
fluorescent biolabels, encapsulating carboxyfluorescein (CF) as
signal amplifiers, that compete with FA for a limited number of
immobilized antibody-binding sites on the stationary phase of an
IAC column (Figure 1). IAC columns are often used to concentrate
analytes; in conjunction with liposomal biolabels, however, they
hold great potential as sensitive immunoaffinity assays for the
determination for FA. We found that the number of liposomes
bound to the anti-FA antibodies was inversely proportional to the
concentration of FA in the sample solution. We observed competi-
tion between the liposomal biolabels and FA for the immobilized
antibody’s binding sites; we quantified the level of FA by
measuring the fluorescence intensity of CF after lysis of the bound
liposomes with elution buffer. The antibody-binding activity was
regenerated after washing with 35% methanol (MeOH) to elute
the bound FA off the column, which we reconditioned by passing
a buffer through the system prior to performing the next
measurement.
EXPERIMENTAL SECTION
Reagents, Materials, and Apparatus. All inorganic chemi-
cals and organic solvents were of analytical grade or the highest
purity commercially available; they were used as received. FA,
dihydrofolic acid, folinic acid (calcium salt), anti-FA monoclonal
antibody (Mab) produced in mouse (clone VP-52, ascites fluid),
cholesterol, 5(6)-carboxyfluorescein, Tween 20, sucrose, tris(hy-
droxymethyl) aminomethane (Trizma base, Tris), dicyclohexyl-
carbodiimide (DCC), octanoic acid, ammonium sulfate, sodium
hydroxide, sodium azide, sodium bicarbonate, potassium dihy-
drogen phosphate, dipotassium hydrogen phosphate, sodium
chloride, sodium acetate, molybdenum blue spray reagent, iso-
propyl ether, chloroform, MeOH, dimethylsulfoxide (DMSO),
pyridine, acetic acid, hydrochloric acid (HCl), and Sephadex G-75-
50 were purchased from Sigma (St. Louis, MO). N-Hydroxysuc-
cinimide (NHS)-activated Sepharose 4 Fast Flow slurry was
purchased from Pharmacia Biotech (Uppsala, Sweden). Dispos-
able polypropylene columns were purchased from Pierce Chemical
(Rockford, IL). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-
N-[amino(polyethylene glycol)2000] ammonium salt (DSPE-
PEG2000-amine), dipalmitoylphosphatidylcholine (DPPC), and
dipalmitoylphosphatidylglycerol (DPPG) were obtained from
Avanti Polar Lipids (Alabaster, AL). Dialysis bags [molecular
(31) Martin, C. A. BNF Nutr. Bull. 1995, 20, 8–15.
(32) Vahteristo, L.; Finglas, P. M. Chromatographic Determination of Folates.
In Modern Chromatographic Analysis of Vitamins; De Leenheer, A. P.;
Lambert, W. E.; Van Bocxlaer, J. F., Eds.; Marcel Dekker: New York, 2000.
(33) Vahteristo, L.; Kariluoto, S. M.; Bärlund, S.; Kärkkäinin, M.; Lamberg-Allardt,
C.; Salovaara, H.; Piironen, V. Eur. J. Nutr. 2002, 41, 271–278.
Figure 1. Schematic representation of the operation of a liposomal immunodiagnostic assay for the detection of folic acid.
weight cutoffs (MWCO): 12 000-14 000 and 6000-8000 Da] were
purchased from Spectrum Laboratories (Rancho Dominguez, CA).
A protein assay kit was purchased from Bio-Rad Laboratories
(Hercules, CA). All solutions were prepared with deionized
water having a resistivity of no less than 18 MΩ cm-1 (Milli-
Q, Bedford, MA). Nuclear magnetic resonance (NMR) spectra
were acquired on a Unity Inova 600 MHz spectrometer (Varian,
Walnut Creek, CA). Mass spectroscopic analysis was performed
by HPLC equipped with an electrospray ionization-mass spec-
trometer (Finnigan LCQ spectrometer, Thermo, San Jose, CA).
The size distribution and ζ potential of the liposomes were
measured using a Brookhaven 90Plus nanoparticle size ana-
lyzer and a ZetaPlus zeta potential analyzer, respectively
(Brookhaven Instruments Corporation, Holtsville, NY).
Distearoylphosphatidylethanolamine-Poly(ethylene gly-
col)2000-Folic acid (DSPE-PEG2000-FA) Bioconjugate. DSPE-
PEG2000-FA bioconjugate was synthesized on the basis of a
modification of methods reported by Gabizon et al.34 and Saul
et al.35 Briefly, DSPE-PEG2000-amine (33.6 mg), DCC (15.3 mg),
and pyridine (167 µL) were added to a solution of FA (16.7
mg) in DMSO (667 mL), and the resulting solution was then
left to react for 4 h at room temperature. After the pyridine
was removed under rotary evaporation, water (4.2 mL) was
added to the solution. The product was dialyzed with a
12 000-14 000 MWCO dialysis bag: twice against 50 mM
sodium chloride (1 L) and three times against water (1 L). The
solution was lyophilized to yield the final product (33.6 mg,
86%). 1H NMR (δ): 0.87 (t, 6H, CH3), 1.25 (s, 56H, CH2),
1.53-1.65 (m, 4H, CH2CH2CO), 2.21-2.39 (m, 8H, CH2CH2CO
and CH2 of Glu), 3.40-3.49 (m, 4H, CH2CH2N), 3.64 (s, ca.
180H, PEG2000), 4.35-4.37 (m, 1H, R-CH), 4.55 (d, 2H, 9-CH2N),
(34) Gabizon, A.; Horowitz, A. T.; Goren, D.; Tzemach, D.; Mandelbaum-Shavit,
F.; Qazen, M. M.; Zalipsky, S. Bioconjugate Chem. 1999, 10, 289–298.
(35) Saul, J. M.; Annapragada, A.; Natarajan, J. V.; Bellamkonda, R. V. J. Controlled
Release 2003, 92, 49–67.
5.20 (m, 1H, PO4CH2CH), 6.64 (d, 2H, 3′,5′-ArH), 7.68 (d, 2H,
2′,6′-ArH), 8.77 (s, 1H, C7-H).
Liposomal Biolabels (FA-Anchored, CF-Encapsulated
Liposomes). The preparation of liposomal biolabels was de-
scribed previously.36,37 In short, aqueous 150 mM CF (1 mL) was
added to a solution of DPPC, cholesterol, DPPG, and DSPE-
PEG2000-folic acid (10:10:1:0.01 molar ratio) in a mixture of
chloroform, isopropyl ether, and MeOH (6:6:1 v/v/v, 4 mL).
After sonication for 5 min, the organic solvent was evaporated
from the mixture under reduced pressure, leaving the lipo-
somes as an orange gel. Another aliquot of CF was added to
this gel, followed by an additional 5 min period of sonication
and vortexing at 45 °C. The liposome size was regulated by
extruding them at least 20 times through 1 and 0.4 µm
polycarbonate filters and then by subjecting them to gel
filtration and dialysis (12 000-14 000 MWCO) to remove any
free, nonencapsulated CF. This suspension of liposomes was
stored in 0.01 M phosphate buffered saline (PBS, pH 7.4)
containing 0.15 M NaCl and 0.12 M sucrose (osmolarity: 430
mmol/kg).
IAC Column. Purified monoclonal anti-FA antibodies were
used to generate the IAC columns. The immunosorbent was
produced on the basis of the modification of the methods reported
by vanSommeren et al. (1993)38 and Ho and Hung (2008).39 NHS-
activated Sepharose 4 Fast Flow slurry was washed on a sintered
glass filter with 1 mM HCl. The gel was then suspended in 0.2 M
NaHCO3 containing 0.5 M NaCl (pH 8.3). The gel solution was
subsequently pipetted into a reaction vial and left to react with
the anti-FA antibody solution for 9 h under shaking (150 rpm,
4 °C). After the coupling reaction was complete, unreacted NHS
groups on the Sepharose medium were blocked through
(36) Ho, J. A. A.; Durst, R. A. Anal. Chim. Acta 2000, 414, 51–60.
(37) Ho, J. A. A.; Hsu, H. W. Anal. Chem. 2003, 75, 4330–4334.
(38) vanSommeren, A. P. G.; Machielsen, P. A. G. M.; Gribnau, T. C. J.
J. Chromatogr. 1993, 639, 23–31.
(39) Ho, J. A. A.; Hung, C. H. Anal. Chem. 2008, 80, 6405–6409.
Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX C
Scheme 1. Synthesis of DSPE-PEG2000-FA
reaction with 0.1 M Tris-HCl (pH 8.5) at 4 °C overnight. The
resulting immunosorbent was then packed into a disposable
polypropylene column (1.9 × 10.7 cm). Noncovalently bound
anti-FA antibodies retained in the IAC column were washed
alternately with three gel volumes of 0.1 M Tris-HCl buffer
(pH 8.5) and three gel volumes of 0.1 M acetate buffer
containing 0.5 M NaCl (pH 4.5) for at least five cycles. The
effluents containing the washed off anti-FA antibody were
collected individually and subjected to protein analysis. The
coupling efficiency was evaluated by monitoring the original
antibody solution (prior to coupling) and the collected effluent
solutions (unbound antibody) spectrophotometrically at 595
nm. Finally, the IAC column was stored at 4 °C in PBS prior
to use.
Real Sample Preparations. Stresstabs, Jamieson, and Health
Diary brands of vitamin B complex tablets were purchased locally;
they contained 400 µg (per 1.00 g tablet), 400 µg (per 0.660 g
tablet), and 800 µg (per 0.400 g tablet) of folic acid, respectively.
After the individual tablets were finely powdered in a porcelain
mortar, a portion of the powder was weighed and then dissolved
in appropriate volumes of 10 mM PBS to form concentrations
corresponding to 10-5 M FA. Each solution was stirred vigor-
ously for 15 min to ensure complete dissolution. The pH of
the tested stock solutions was adjusted to pH 7-8 and were
stored at -20 °C prior to use. The working solutions were
prepared through 100-fold dilution of the stock solutions to form
concentrations corresponding to 10-7 M FA.
Assay Procedure. The assay procedure was initiated by
equilibrating the IAC column with 10 mM phosphate buffer (pH
7.4) containing 0.15 M NaCl and 0.12 M sucrose. The FA sample
(500 µL) was loaded onto the IAC column and then rinsed with
the same buffer to wash off any unbound FA. The liposome
solution (500 µL) was introduced to occupy the remaining available
antibody binding sites. The IAC column was rinsed again to flush
out any excess unbound liposomes. The elution buffer (35% MeOH
D Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX
in Tris buffered saline (TBS) containing 0.1% Tween 20) was then
passed through the column, causing the rupture of liposomes and
the release of the encapsulated CF dye molecules. A portion of
the effluent (360 µL) was collected, and its fluorescence intensity
was measured spectrophotometrically (excitation, 494 nm; emis-
sion, 517 nm). Finally, the antibody’s binding sites were regener-
ated through continuous flushing of the column with the elution
buffer. The column was reconditioned with PBS prior to subse-
quent analyses.
RESULTS AND DISCUSSION
Characterization of DSPE-PEG2000-FA. Scheme 1 depicts
our synthetic approach toward DSPE-PEG2000-FA. We performed
thin-layer chromatography (TLC) analysis on 10 × 20 cm silica
gel plates, eluting with CHCl3/MeOH/H2O (50:50:6, v/v/v).
A new spot having an Rf of 0.97, due to DSPE-PEG2000-FA,
appeared under UV examination (the corresponding Rf of folic
acid was 0). We confirmed the presence of phospholipids by
resolving the plate with a molybdenum blue spray, which
revealed blue spots having Rf values of 0.90 and 0.97 for DSPE-
PEG2000-amine and DSPE-PEG2000-FA, respectively.
The 1H NMR spectrum of DSPE-PEG2000-FA revealed signals
at 0.87 and 8.77 that are characteristic of its methyl group on
DSPE and of the proton on the pteridine ring of folic acid,
confirming the successful conjugation of DSPE-PEG2000 to folic
acid.
LC/MS analysis of DSPE-PEG2000-FA revealed bell-shaped
distributions at m/z 1088.3 (charge ) +3) and m/z 1631.7 (charge
) +2), suggesting that the molecular weight of the product was
ca. 3264 Da. This molecular mass is consistent with the proposed
structure.
Characterization of Liposome Biolabels. The size homo-
geneity of the liposomal biolabels is the key aspect affecting the
development of reproducible and reliable liposome-based diag-
nostic applications. In this study, we extruded the liposome
Figure 2. Fluorescence signals (λex, 490 nm; λem, 520 nm) generated
at various volumes of added liposome biolabels.
preparations through polycarbonate filters to decrease their size
heterogeneity. Using a nanoparticle size analyzer, we determined
an average diameter for the liposomes of 249 ± 37 nm, suggesting
that the average volume of a single liposome was ca. 8.1 × 10-12
µL, with an entrapped volume (assuming a bilayer thickness
of 4 nm)40 of ca. 7.3 × 10-12 µL. Assuming that the CF
concentration inside the liposomes was equal to that in the
original solution and comparing the fluorescence of the lysed
liposomes with that of standard CF solutions, we calculated
that there were ca. 9.4 × 1012 liposomes/mL and that each
liposome contained 6.6 × 105 molecules of CF. Because the
average surface areas of DPPC and cholesterol molecules are
ca. 71 and 19 Å2, respectively,40 we estimated that ca. 103
molecules of FA were present on the outer surface of each
liposome, given that 0.005 mol % of DSPE-PEG2000-FA was
successfully incorporated during the formation of liposomes.
The average ζ potential of the liposomal biolabels was -11.86
± 0.76 mV, consistent with the existence of negatively charged
DPPG units in the liposome structure.
Optimization of Parameters. Figure 2 depicts the effect of
the amount of liposome on the performance of the IAC assay for
FA. We diluted various volumes of the liposome concentrate to
500 µL and found that the optimal added amount was 100 µL;
this sample contained ca. 9.4 × 1011 liposomes and encapsulated
ca. 1.0 × 10-6 mol of CF dye. We also investigated the optimal
amount of anti-FA antibody for use in the manufacture of the
IAC column. Accordingly, we manufactured IAC columns
containing 0.1 and 0.4 mg of anti-FA antibody, respectively.
Figure 3 reveals a much more noticeable competition between 0
and 10-3 M FA (analyte) solutions when using the IAC column
incorporating 0.4 mg of the anti-FA antibody. This IAC column
offered a minimal, but sufficient, number of antibody binding
sites; we chose it for our subsequent studies. In contrast, we
observed poorer competition when using the column incorpo-
rating 0.1 mg of the antibody, presumably because of the
paucity of antibody binding sites.
Table 1 lists the coupling efficiencies of the two IAC columns.
We calculated the amount of anti-FA antibody coupled by
(40) Israelachvili, J. N.; Mitchell, D. J. Biochim. Biophys. Acta 1975, 389, 13–
19.
Figure 3. Effect of the amount of anti-FA antibody used to prepare
IAC columns on the competition between FA-anchored liposomal
biolabels and FA.
subtracting the total amount of anti-FA found in all of the wash
fractions from the amount of anti-FA offered. The coupling
efficiency was determined by measuring values of A595, using a
Bio-Rad protein assay, based on the method of Bradford.41 We
observed almost no residual anti-FA antibody in the wash
fractions, indicating almost quantitative coupling.
Assay Performance. For each analysis, we loaded 500 µL of
FA onto the IAC column. These FA molecules bound to the
antibodies, occupying a fraction of the total number of antibody
binding sites in proportion to their concentration. An aliquot of
liposomes (500 µL, appropriately diluted) was then added onto
the column, resulting in binding to the unoccupied binding sites
of the antibody.
We transformed the collected data into a binding ratio using
the equation
binding ratio ) F/F0 × 100%
where F is the fluorescence signal generated at a given concentra-
tion of FA and F0 is the fluorescence signal in the absence of
FA. For our IAC column prepared with 0.4 mg of the anti-FA
antibodies, Figure 4 displays the dose-response curve that we
obtained after plotting F/F0 (%) against the concentration of the
FA standards. The inset reveals a correlation coefficient (R2)
of 0.9853 for the linearized portion of the curve between 10-8
and 10-4 M (a wide dynamic range of at least 4 orders of
magnitude). The standard curve was, therefore, fitted to a four-
parameter logistic equation according to the formula y ) {(A2
- A1)/[1 + 10 exp((log (X0 - X)) × p)]} + A1, where A1 and
A2 are the maximal and minimal fluorescence signals, respec-
tively, while X0 is the concentration producing half of the
maximal fluorescence signal, X is the FA concentration, and p
is the slope at the inflection point of the sigmoid curve.27 We
define the operational detection limit after subtracting 3 times
the standard deviation of the control (free of FA) from its
(41) Bradford, M. M. Anal. Biochem. 1976, 72 (1-2), 248–254.
Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX E
Table 1. Efficiency of Coupling Anti-FA Monoclonal Antibodies to Sepharose Gel Beads

coupling efficiency
amount of anti-FA amount of unbound anti-FA amount of anti-FA (amount of anti-FA coupled/
offered (mg) (from protein assay; mg) coupled (mg) amount of anti-FA offered; %)
0.10 0 0.10 100
0.40 0 0.40 100

average value. Accordingly, the limit of detection (LOD) for

FA detected by the IAC column was 3.1 × 10-8 M with a 99.7%
level of confidence.
Our elution buffer (35% MeOH in TBS containing 0.1% Tween
20) was capable not only of releasing the liposomal CF dye
molecules but also of regenerating the antibody’s activity, which
allowed the immunoreactor to be used for at least 50 sample
injections without any observable loss of reactivity (data not
shown).
Cross-Reactivity of Anti-FA Mab-Containing IAC Column.
Figure 5 reveals that the anti-FA Mab IAC column did not display
significant cross-reactivity toward either of the FA derivatives
dihydrofolic acid or folinic acid (calcium salt). After loading
solutions (10-4 M) of FA, dihydrofolic acid, and folinic acid
(calcium salt) into the IAC column and defining the binding
reactivity of FA toward anti-FA Mab as 100%, we determined
that the binding reactivities of these FA derivatives toward anti-
FA Mab on the Sepharose beads were 3.1 and 3.7%, respectively.
Real Sample Analysis. We analyzed real samples to investi-
gate the performance our IAC assays for the determination of FA.
Thus, we analyzed samples from three brands of vitamin B
complex tablets (Stresstabs, Jamieson, and Health Diary) for their
FA contents. We diluted these samples to desired concentrations
prior to analysis so that they would fit into the linear portion of
our dose-response curve. We then loaded 500 µL of each sample
solution directly onto the IAC column and performed the assay
using the same procedure as described above for the stock
solutions. Table 2 summarizes the quantitative data obtained from
the IAC; the FA contents correlate well with the values listed on
Table 2. Recoveries of FA from the IAC Analyses of
Real Samples
labeled amount found amount recovery
vitamin brand (µg/tablet) (µg) (found/labeled; %)
Stresstabs 400 449 ± 62 112
Health Diary 800 764 ± 72 95.5
Jamieson 400 362 ± 36 90.5
the nutrition facts labels provided with each vitamin brand. Less
than 30 min is needed to run a single assay. Therefore, our IAC
assay appears to be suitable for the rapid and simple analysis of
real samples without the need for complicated sample precon-
centration or purification techniques.
CONCLUSION
We have developed a simple, yet sensitive, immunodiagnostic
column assay for the determination of an important water-soluble
vitamin, FA, via liposomal amplification. The combination of CF-
encapsulated, FA-anchored, PEGgylated liposomes and an IAC
column packed with anti-FA Mab-immobilized Sepharose beads
enables the construction of an IAC for determining FA levels in
real samples. The use of fluorescent liposomal biolabels amplified
the signals when testing low concentration of FA; in addition, the
IAC column served as a concentrator to extract FA efficiently from
the sample solutions. This approach offers acceptable sensitivity
[LOD for FA: 6.8 ng (equivalent to 500 µL of 3.1 × 10-8 M FA)],
comparable with those reported previously, with a wide
dynamic range (4 orders of magnitude). With its low LOD, high
accuracy, and short analysis times (<30 min), this optimized
liposome-based IAC assay should allow the user-friendly
surveillance of FA levels in foodstuffs, infant formulas, feeds,

Figure 4. Dose-response curves obtained from IAC columns coated

with 0.4 mg of anti-FA; error bars, (1 standard deviation. Sensitivity
is defined as the capability of responding reliably and measurably to
changes in folic acid concentration and is represented as the slope Figure 5. Plot of the binding activity of each folate derivative on the
of calibration curve. Inset: linear portion of the main curve. IAC column with respect to that of FA.

F Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX

and nutritional supplements. Our IAC assay possesses several folic acid-sensitized liposomes, and development of the immuno-
advantageous features when compared with chromatographic diagnostic immunoassay. M.-Y. Liao is responsible for the NMR
or MBA methods, including more rapid sample preparation spectroscopic analysis. L.-C. Wu is responsible for the MS analysis.
(sample preconcentration and analysis are performed concur- This study was supported by the National Science Council in
rently), increased analytical turnover rate, and savings in labor Taiwan under Grants NSC 95-2113-M-007-040-MY3 and 97-2113-
and time (no washing or preincubation were needed) because M-260-006-MY2.
of simple sampling and cleanup of samples prior to each
analysis. Furthermore, the sensitivity and assay time of our
SUPPORTING INFORMATION AVAILABLE
IAC assay is comparable to the electrochemical magneto
Purification of mouse anti-FA Mab.42 This material is available
sensors recently developed by Lermo et al. (2009).27 In
free of charge via the Internet at http://pubs.acs.org.
conclusion, the acceptably sensitive LOD and high assay speed
and the cost-effectiveness, reusability, and portability of the
method suggest that this proposed system may be extended
Received for review February 23, 2009. Accepted May 19,
to the monitoring and surveillance of various vitamins.
2009.

ACKNOWLEDGMENT AC900402V
J.-a. A. Ho’s group is responsible for synthesis of folic acid-
anchored PEGgylated phospholipid bioconjugate, preparation of (42) Mckinney, M. M.; Parkinson, A. J. Immunol. Methods 1987, 96, 271–278.

Analytical Chemistry, Vol. xxx, No. xx, Month XX, XXXX G