Spectrochimica Acta Part A 86 (2012) 456–460

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Determination of l-phenylalanine on-line based on molecularly imprinted

polymeric microspheres and flow injection chemiluminescence
Huamin Qiu, Yulei Xi, Fuguang Lu, Lulu Fan, Chuannan Luo ∗
School of Chemistry and Chemical Engineering, University of Jinan, No. 106 Jiwei Road, Jinan 250022, China

a r t i c l e i n f o a b s t r a c t

Article history: A novel molecular imprinting-chemiluminescence (MIP-CL) sensor for the determination of l-
Received 30 August 2011 phenylalanine (Phe) using molecularly imprinted polymer (MIP) as recognition element is reported. The
Received in revised form 20 October 2011 Phe-MIP was synthesized using acrylamide (AM) as functional monomer and ethylene glycol dimethacry-
Accepted 29 October 2011
late (EGDMA) as cross-linker, 2,2-azobisisobutyronitrile (AIBN) as initiator and the polymers’ properties
were characterized. Then the synthesized MIP was employed as recognition element by packing into flow
Keywords:
cell to establish a novel flow injection CL sensor. The CL intensity responded linearly to the concentration
Molecularly imprinted polymeric
of Phe in the range 1.3 × 10−6 to 5.44 × 10−4 mol/L with a detection limit of 6.23 × 10−7 mol/L (3), which
microspheres
Flow injection
is lower than that of conventional methods. The sensor is reusable and has a great improvement in sen-
Chemiluminescence sitivity and selectivity for CL analysis. As a result, the new MIP-CL sensor had been successfully applied
Phenylalanine to the determination of Phe in samples.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction linear range and simple equipment. It is used widely in water

The aromatic amino acid l-phenylalanine (Phe) is used in
healthy, pharmaceutical and food products [1,2]. Individual sep-
aration and determination of Phe from the co-existing amino acids
in the microbial processes and from the substrates in the enzymatic
processes are necessary for its subsequent processing. Numerous
studies on the separation of Phe from other amino acids for ana-
lytical purposes have been published. According to the previous
report, the methods used in determination of Phe were phenylala-
nine oxidase [3], electrochemical chromatography [4] and liquid
chromatography [5]. Those methods can offer accurate determina-
tion results. However, some of them need expensive equipments
and complex procedures for sample pretreatment, while the other
suffers from low selectivity.
FI-CL has caused considerable attention for its convenient oper-
ation, rapid determination and accuracy. According to the linear
relationship between CL intensity and concentration of solution
[6], FI-CL method has many advantages in the determination of
samples as an analysis method, such as high sensitivity, wide
∗ Corresponding author. Tel.: +86 531 89736065.
E-mail address: chm luocn@ujn.edu.cn (C. Luo).
quality testing [7], soil sample analysis, agriculture and environ-
mental monitoring [8], drug research [9], blood analysis [10] and
food analysis [11]. But the method was limited in development
and application for poor selectivity. Molecularly imprinted tech-
nology was introduced into CL for improving selectivity which has
high specificity recognition [12]. To produce molecularly imprinted
polymers, two different approaches have been developed: the
set-up of covalent interactions between template molecules and
monomers [13] and the development of reversible non-covalent
interactions (mainly hydrogen bonding) between them [14,15]. The
molecularly imprinted materials have been applied into affinity
separation [16], antibody binding [17], biomimetic chloroperox-
idase [18] and biomimetic sensors [19], which were excellently
examined in recent developments and perspective area evolutions
such as chromatographic stationary phase [20], solid phase extrac-
tion matrices [21,22], artificial receptors [23], antibody mimics
[24], enzyme mimics [25], catalytic application [26], recognition
elements in sensors [27] and CL sensing systems [28].
In this paper, a novel MIP-CL sensor for Phe determination
using MIP as recognition element is developed. Phe-MIP can be
prepared using acrylamide (AM) as functional monomer and ethy-
lene glycol dimethacrylate (EGDMA) as cross-linker. Particles of
the resultant MIP were packed into a piece of a straight shape

1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.10.068
 H. Qiu et al. / Spectrochimica Acta Part A 86 (2012) 456–460 457

Fig. 1. The schematic diagram of the MIP-CL sensor system.

Fig. 2. The schematic diagram of preparation.

glass tube, which served as a flow cell positioned in front of 2. Experimental

a FI-CL analyzer. Phe is selectively adsorbed on the MIP on-
line, reacting with the alkaline luminol–H2 O2 to produce strong
post-chemiluminescence (CL). After the CL reaction, the absorbed
Phe was destroyed and removed by the flow solution with cav-
ities left on the MIP ready for the next adsorption assay. The
reusable sensor was successfully used in determining Phe in milk
samples.
2.1. Materials and reagents
Phe were supplied by Aladdin Reagent Co., Ltd (China); AM, AIBN
and EGDMA were supplied from Sigma–Aldrich; the EGDMA and
AM were distilled to remove inhibitors. AIBN was re-crystallized
prior to its use.

Fig. 3. The flow chart of measuring samples.

458 H. Qiu et al. / Spectrochimica Acta Part A 86 (2012) 456–460

The Phe stock solution (1.0 × 10−2 mol/L) and luminal stock
solution (1.0 × 10−2 mol/L) were prepared and stored in refrig-
erator, respectively. The methanol, acetone, acetic acid, sodium
hydroxide and all the other chemicals used were of analytical
reagent grade obtained from Tianjin Chemical Co., Ltd. (China).
Doubly distilled water was used throughout the work.

2.2. Apparatus

The schematic diagram of flow system used in this study was

shown in Fig. 1 [29]. All solutions were delivered through two peri-
staltic pumps. PTFE tubing (0.8 mm i.d.) was used to connect all
components in the flow injection CL analyzer (Xi’an Remex Elec-
tronic High-Tech, Ltd., China). The data acquisition and treatment
were performed with the IFFM-D flow injection CL data processing
software (Xi’an Remex Electronic High-Tech, Ltd., China).

2.3. Phe-MIP preparation

Fig. 4. The choice of functional monomer: (a) blank; (b) methacrylic acid; (c) acry-
Two imprinted and non-imprinted molecular polymeric micro- lamide; (d) the mixture of methacrylic acid, acrylamide.

spheres were prepared using Phe as template, AM as functional

monomer and EGDMA [30] as common cross-linker. A typical
the NIP-cell and Phe molecules in the sample solution cannot selec-
preparation of the molecularly imprinted polymers was carried
tively adsorb on the polymer.
out as follows [31]. A 25 mL ethanol solution containing 0.1 mmol
Phe and 0.4 mmol AM was prepared. After degassing and nitrogen
purging for about 5 min to remove oxygen which inhibits the poly- 2.5.4. Chemiluminescence detection sample solution with Phe
merization, the solution was placed at 25 ◦ C and reacted for 12 h. In this step, assistant pump and main pump were both started
Then 2 mmol EGDMA and 20 mg AIBN were added into the solu- and switch valve was in connection with 1. The merged stream of
tion which was continued for 24 h at 60 ◦ C. The polymers were then luminol, hydrogen peroxide and sodium hydroxide flowed to react
extracted with methanol/acetic acid (9/1, v/v) [32] overnight and with sample solution with Phe to produce CL2 .
washed with distilled water to remove any non-grafted polymer,
monomer, residual initiator and the template. The non-imprinted
2.5.5. Cleaning the MIP-cell and NIP-cell for reuse
molecular polymeric microspheres were prepared and processed
In this step, main pump was stopped and switch valve was
in the same way, but in the absence of any template. Fig. 2 shows
in connection with 1 and 2; eluent and ultrapure water flowed
the schematic diagram and the surface morphology was shown in
through the MIP-cell to remove Phe for next determination.
Fig. 5.

2.4. MIP flow cell make-up 2.5.6. Calculation the concentration of Phe
I = CL2 − CL1 , according to regression equation, the concentra-
50 mg MIP and 50 mg NIP particles were packed into straight tion of Phe was obtained.
shape glass tube, respectively, which were served as flow cell. Both
ends of the tube were stuffed with a small amount of glass wool
3. Results and discussion
[28]. The tube was connected with the flow system. The MIP flow
cell must be conditioned prior to its first time use.
3.1. The choice of functional monomer

2.5. MIP-CL sensors measuring samples

After the reaction of template molecule and functional
monomer, UV spectrophotometry was used for choosing functional
The schematic diagram for flow-through MIP-CL sensor was
monomer in methacrylic acid and acrylamide and the mixture of
shown in Fig. 1 and the determination could be summarized as
methacrylic acid and acrylamide [34], as shown in Fig. 4.
five steps shown in Fig. 3 [33].
With the addition of functional monomer, red shift appeared
which was the maximum absorption wavelength of Phe. As the
2.5.1. Recognition and adsorption of Phe
result shows, the red shift of Phe and acrylamide is the largest;
In this step, assistant pump was operated and switch valve was
acrylamide was chosen as best functional monomer for forming
in connection with 1, sample solution was delivered to flow through
more hydrogen bonds with Phe. The reason was that, O and NH2 –
the MIP-cell and Phe molecules in the sample solution were selec-
of acrylamide can form hydrogen bonds, which as recognition sites,
tively adsorbed on the polymer.
but in methacrylic acid, only O .

2.5.2. Chemiluminescence detection sample solution without Phe

In this step, assistant pump and main pump were both started
and switch valve was in connection with 1. The merged stream of
luminol, hydrogen peroxide and sodium hydroxide flowed to react
with sample solution without Phe to produce CL1 .
2.5.3. Transit of Phe
In this step, main pump was operated and switch valve was in
connection with 2, sample solution was delivered to flow through
3.2. The SEM photographs of the MIP
SEM specimens were prepared by redispersing the micro-
spheres in ethanol and placing a drop on a piece of cover glass,
which was mounted on an aluminum stud. After solvent evapora-
tion, the particles were sputter-coated with a thin layer of gold [35].
Morphology of MIP was obtained as shown in Fig. 5. As the results
shows, the MIP was almost of the same nanometer level.
 H. Qiu et al. / Spectrochimica Acta Part A 86 (2012) 456–460
Fig. 5. ESM of MIP.
Fig. 6. Infrared Spectrogram of NIP and MIP.
3.3. Infrared spectrogram of NIP and MIP
As can be seen, the telescopic vibration peak of –NH2 :
3300–3500 cm−1 and the vibration peak (hydrogen bonding) of
–COOH: 2500–3000 cm−1 were obtained from Fig. 5 which was the
characteristic peak of Phe [36]. Compared to NIP, the characteristic
peak of Phe in MIP has an obvious displacement effected by hydro-
gen bonding. The results proved that hydrogen bonding existed in
synthesized MIP [37] (Fig. 6).
3.4. Optimization of MIP-CL sensor
As it is shown from the experiment results, weak CL emis-
sion was observed when the reaction between luminol and H2 O2
occurred in isolation; however, when Phe solution was added, the
CL emission remarkably increased. The peak heights of the CL emis-
sion were proportional to the concentration of Phe.
The optimization experiment was carried out to get a bet-
ter knowledge of the CL reaction of luminol–H2 O2 –Phe through
the schematic diagram shown in Fig. 1. With the luminol con-
centration in the range from 4.0 × 10−4 to 1.2 × 10−3 mol/L, the
459
Fig. 7. The regression equation.
CL intensity increased with raising concentration of luminol
up to 6.4 × 10−4 mol/L. Above 6.4 × 10−4 mol/L the CL intensity
decreased. Thus, the 6.4 × 10−4 mol/L luminol was chosen for fur-
ther work.
The effect of H2 O2 concentration was examined from 0.06 to
0.28 mol/L, the CL intensity reached maximum when H2 O2 was
0.18 mol/L. In fact, a final optimum concentration of 0.18 mol/L was
selected in the following work.
The effect of NaOH concentration as a medium was exam-
ined over 0.008–0.016 mol/L range and the CL intensity reached
maximum value when 0.024 mol/L NaOH was used. But higher
concentration of NaOH lowered the CL intensity of this system.
The concentration of NaOH used in the next experimental was
0.024 mol/L.
The adsorption time which depends on peristaltic pumps speed
is an important parameter for the amount of Phe absorbed on the
MIP. The effect of pumps speed on the biosensor is investigated
through changing the speed, and the relation of CL intensity with
the pumps speed was observed under the condition of 10–50 r/min.
It was observed that the adsorption reached maximum at 20 r/min.
Simultaneously, according to experiment results, it was seen that
other substances would be adsorbed by MIP when the pumps speed
was too small. Phe molecules cannot adsorb by MIP completely
under too large pumps speed. So the 20 r/min pumps speed was
chosen throughout the entire study [38].
3.5. The analytical performance of MIP-CL sensor for the
determination of Phe
Under optimal conditions, the CL intensity responded lin-
early to the concentration of Phe in the range from 1.3 × 10−6
to 5.44 × 10−4 mol/L with a detection limit of 6.23 × 10−7 mol/L
(3), which is much lower than conventional methods [39,40].
The regression equation is ICL = 2.18 × 103 + 1.83 × 103 c (c being the
Phe concentration (mol/L)) with a correlation coefficient of 0.9948
as shown in Fig. 7. The relative standard deviation (RSD) for the
determination of 3.0 × 10−4 mol/L Phe was less than 3.0% (n = 11).
3.6. Interferences study
Under the optimum conditions, the factors which may interfere
potentially the determination of Phe were investigated. The relative
error was controlled within ±5.00% and the tolerance times were
as shown in Fig. 8.
These results showed that MIP can be used as recognition mate-
rial in the CL analysis and improve the selectivity of the CL method.
Because the contents of coexisting substances were all lower than
their tolerable concentrations, the proposed sensor could be used
directly to determine the Phe in milk samples.
460 H. Qiu et al. / Spectrochimica Acta Part A 86 (2012) 456–460

Table 1
Results of recovery tests (n = 6).

Samples Adding (10−4 g mL−1 ) Phe content (10−4 g mL−1 ) Found (10−4 g mL−1 ) Recovery (%) RSD (%)

1# 2 87.4 2.02 101.0 1.83

2# 4 86.9 3.99 99.75 3.49
3# 8 87.5 8.23 102.8 2.96

due to loss of binding sites; however, it was easy to replace the

MIP-cell in the channel.

4. Conclusions

In this work, the Phe-MIP was used as molecule recognition

material in the CL analysis. The characteristic of the selective bind-
ing function of Phe-MIP to Phe molecule enables the method to
have the advantage of selectivity, making it possible to be applied
to analysis of Phe in milk samples directly. The application of the
method was validated by testing the recovery of known amount
of Phe in milk samples. The sensor was successfully applied to the
determination of Phe in milk samples with satisfactory results. And
the obtained MIP-CL sensor has shown to provide a sensitive and
fast method for on-site determination of Phe.

Fig. 8. Tolerable ratio of interfering species to Phe with and without MIP: 1,
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