1. Sensors and Actuators B 240 (2017) 631–639
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical
journal homepage: www.elsevier.com/locate/snb
Ultra-trace analysis of d-and l-aspartic acid applying one-by-one
approach on a dual imprinted electrochemical sensor
Bhim Bali Prasad∗
, Swadha Jaiswal, Kislay Singh
Analytical Division, Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, India
a r t i c l e i n f o
Article history:
Received 13 July 2016
Received in revised form 23 August 2016
Accepted 7 September 2016
Available online 9 September 2016
Keywords:
Dual imprinted polymer
Multi-walled carbon nanotubes
Gold nanoparticles
d- and l-Aspartic acid
Potassium ferricyanide probe
One-by-one approach
a b s t r a c t
An enantiomeric pair imprinted polymeric film, with embedded functionalized multiwalled carbon nano-
tubes, was developed following the ‘surface grafting from’ approach onto a gold nanoparticles decorated
pencil graphite electrode. Double imprinting of chiral molecules in a single polymer motif, as a sensory
platform, for the one-by-one evaluation of individual components of a racemic mixture, is a challenging
task. The underlying state-of-art proposed for this purpose is novel and if this works well, any two simple
molecules could be used as templates. In this work, a pencil graphite electrode was first dipped for the
overnight in the aqueous suspension of gold nanoparticles. The electrode was then subjected to spin
coating with a pre-polymerization mixture consisting a monomer (N-acryloyl-pyrrolidine-2,5-dione),
templates (d- and l-Aspartic acid), a cross-linker (ethylene glycol dimethacrylate) in the presence of an
initiator (␣,␣ -azoisobutyronitrile). As aspartic acid isomers have been found to be electro-inactive, their
evaluation was feasible indirectly with the help of a potassium ferricyanide probe. The quantitation abil-
ity of the proposed sensor, with differential pulse anodic stripping voltammetric transduction, was found
to be in the tune of 4.08 ng mL−1
(S/N = 3) for both the enantiomers in the real samples, which reportedly
manifest several chronic diseases at their stringent limits.
© 2016 Published by Elsevier B.V.
1. Introduction
In nature, chirality of a molecule is as important as its chemical
makeup. Our body recognizes chirality in terms of their distinct
pharmacological profiles. In many cases, one enantiomer is an
active pharmaceutical ingredient while the other can be benign
or even toxic. Apart from their difficult isolation, the quantitative
analysis of enantiomers is a major issue from the standpoint of effi-
cacy and safety of drugs [1]. Although the most prevalent optical
isomers of amino acids are having L-configuration, the occurrence
of D-configuration has also been found in human, both in the free
form and bound in peptides or proteins, in high concentrations to
fulfil specific biological function.
Aspartic acid (Asp), being a non-essential amino acid, is a
major excitatory neurotransmitter in the central nervous system. d-
aspartic acid (d-Asp) serves in the central brain region to cause the
release of hormones, viz., luteinizing hormone, follicle-stimulating
hormone, growth hormone, and sex hormone. According to the lit-
erature [2], Asp concentration levels in cerebrospinal fluid (CSF)
could be correlated with a number of neurological disorders such as
∗ Corresponding author.
E-mail address: prof.bbpd@yahoo.com (B.B. Prasad).
Alzheimer’s (7.19 ± 2.53 ng mL−1), epilepsy (4.22 ± 2.40 ng mL−1),
and lennox syndrome (4.21 ± 1.99 ng mL−1). In particular, l-Asp
can be a biomarker for lung cancer and head and neck can-
cer manifested at 266 ng mL−1 and 930 ng mL−1, respectively in
serum. Hence, analysis of d & l-Asp in CSF and serum may
provide a suitable mean of diagnosis and possible treatment of
neuropsychiatric diseases. Numerous methods for the enantiose-
lective analysis of d- or l-Asp have been reported namely, thin
layer chromatography [3] gas chromatography [4] high perfor-
mance liquid chromatography [5], chiral ligand-exchange capillary
electrophoresis [6], spectrophotometry [7], and fluorometry [8].
However, these methods are time-consuming, solvent-usage inten-
sive, and highly expensive. Although electrochemical detection can
be considered relatively very simple and elegant, it has been failed
so far to reveal acute Asp levels that can cause the prognosis of
epilepsy and lennox syndrome at primitive stage [2]. Thus, a highly
selective and sensitive electrochemical system for Asp evaluation
is called for.
Simply put, molecularly imprinted polymers (MIPs) are syn-
thetic receptors made with the signature of template(s) by
arranging suitable monomer(s) around it, subsequently followed
by polymerization in the presence of a cross-linker. The retrieval
of template molecules generates molecular cavities in the poly-
mer network, which are specific for the molecular recognition
http://dx.doi.org/10.1016/j.snb.2016.09.031
0925-4005/© 2016 Published by Elsevier B.V.

2. 632 B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639
in terms of size and shape of the template. Molecular architec-
ture in the imprinting world witnessed a very limited attention
for imprinting two or more targets (print molecules) in a single
MIP format [9–18]. Memory sites for only a single compound in
MIP network might exert restriction in the detection of a group
of analytes present in samples. Although multi imprinting saves
both time and labor as compared to the traditional imprinting,
the diffusion pathways of templates are often crisscrossed and
disturbed during their recapture and/or stripping processes. This
could be the reason that no attempt has been made so far for
the simultaneous/sequential analysis of chiral compounds. How-
ever, with the advent of nanotechnology; an ultra thin layer of
MIP on a solid substrate might improve the analyte diffusitivity
uninterruptedly. Such nano-structured materials can be exploited
as sensors to exhibit a high degree of success in the improve-
ment of detection sensitivity and selectivity. Molecularly imprinted
electrochemical sensors apparently combine the characteristics
of electrochemical detection and molecular imprinting technol-
ogy. The underlying state-of-art in fabricating a nanosensor in the
present work is typical in the sense that we have initially used gold
nanoparticles (AuNPs) decorated pencil graphite electrode (PGE)
for the subsequent surface modification with multiwalled car-
bon nanotubes (MWCNTs) interspersed doubly imprinted polymer,
employing the ‘surface grafting from’ approach. Herein, the embed-
ded CNTs have advantages of enhanced electronic properties and a
large edge plane/basal plane ratio to impart rapid electrode kinet-
ics. On the other hand, AuNPs would effectively serve as “electron
antennae” for channelling electron transport between the electrode
and the electro-active species. Moreover, MWCNTs embedded thin
polymer film artificially anchored onto AuNPs/PGE, in physical or
chemical manner, may show much potentiality for the effective
mass transport with a facilitated egress-ingress of the test analyte,
under the pool of imprinting and electrostatic effects [19].
Double imprinting in itself is an arduous task with the appre-
hension of its success in the field of molecular recognition.
Despite the fact that chiral compounds exists in two enantiomeric
forms with the identical molecular formula, their different spa-
tial conformations forming non-superimposable mirror images
are advantageous to generate distinct D-specific and L-specific
molecularly imprinted cavities, for enantioselective recognition.
Nevertheless, the voltammetric peak separation of D- and L-
isomers is not possible as a consequence of their identical redox
behaviour. The situation may further be critical when both isomers
are electro-inactive in nature. In the present work, the model pair
of analytes, d-Asp and l-Asp, selected for analysis is found to be
electrochemically inactive. We have, thus, endeavoured for the first
time to resolve this problem by developing a protocol for double
imprinting of d-Asp and l-Asp in a single polymer motif, as out-
lined in Scheme 1. In this work, we have proposed a novel method
of indirect enantioselective analysis of d- and l-Asp, with the help of
a ferricyanide probe, using a single modified electrochemical sen-
sor. Accordingly, one has to saturate both of the imprinted cavities
with the probe molecules and initially obtain the corresponding
differential pulse anodic stripping voltammetry (DPASV) signal in
phosphate buffer (pH = 3). After washing the electrode with water,
the magnitude of probe response remains unaltered. Further, for
the quantification of d-Asp, this electrode is exposed to l-Asp by
replacing the probe solution from corresponding cavities till sat-
uration. The D-isomer cavities duly filled-in with probe solution
are now all set for the quantitative analysis of d-Asp. The elec-
trode at this stage is regenerated by extraction of templates from
both type of cavities. Similar procedure is adopted for the anal-
ysis of other isomer, l-Asp, using the same refreshed electrode.
Despite the fact that MIP-sensors for enantioselective analysis of
individual isomers, hitherto, have been utilized two separate elec-
trodes [2,20,21], the proposed method of one-by-one sensing on
a single MIP-sensor could relatively be more sensitive, accurate
and cost-effective. Furthermore, this sequential method of indirect
analysis can be extended to the determination of any pair of simple
molecules, whether electro-active or electro-inactive in nature.
2. Experimental
2.1. Chemicals and reagents
All chemicals were of analytical reagent grade, and used without
further purification. Demineralized triple distilled water (con-
ducting range 0.06–0.07 × 10−6 S cm−1) was used throughout the
experiment. Succinimide (pyrrolidine-2,5-dione), acryloyl chlo-
ride, potassium ferricyanide, trisodium citrate, and chloroauric acid
(HAuCl4·H2O) were purchased from Loba Chemie (Mumbai, India).
All solvents, dimethylsulphoxide (DMSO), tetrahydrofuran (THF),
triethylamine (TEA), and ethanol, were procured from Spectrochem
Pvt. Ltd. (Mumbai, India). Ethylene glycol dimethacrylate (EGDMA),
MWCNTs (internal diameter 2–6 nm, outer diameter 10–15 nm,
length 0.2–10 ␮m, and purity >90%), ␣,␣ -azoisobutyronitrile
(AIBN), d-Asp, and l-Asp were obtained from Aldrich (Steinheim,
Germany). All interferents studied were purchased from Fluka
(Steinheim, Germany). The supporting electrolyte used was phos-
phate buffer solution (pH 3.0, ionic strength 0.01 M). Standard
stock solutions of d-Asp and l-Asp (500 ␮g mL−1) and potassium
ferricyanide (33 ␮g mL−1) were prepared in water. All working
solutions were prepared by diluting stock solution with water.
Human blood serum and CSF were obtained from the Institute of
Medical Science, Banaras Hindu University (Varanasi, India) and
kept in refrigerator below −4 ◦C, before use. The pharmaceutical
sample analyzed was astymin hepa (Tablets India Ltd., Chennai,
India). Pencil rods (2B grade, 2 mm diameter, 5.0 cm length) were
purchased from Hi Par, Camlin Ltd. (Mumbai, India). The PGE
was used for modification with MIP because it is better in terms
of higher electrochemical activity, commercial availability, good
mechanical stability, low cost, low background current, and wide
potential window as compared to other solid electrodes (Pt, Au, Pd,
Ag, glassy carbon, etc.) [22,23]. Furthermore, PGE possesses several
graphite pores into which MIP film could be physically adsorbed,
with firm adherence as compared to glassy carbon electrode and
other solid electrodes.
2.2. Apparatus
DPASV and cyclic voltammetry (CV) were performed on a
portable potentiostat ␮-Stat 200 (Drop Sens S.L. Oviedo, Spain),
which was connected via USB to a computer with measurement
software Drop View (DropSens). The electrochemical cell was con-
sisted of MIP-AuNPs@PGE, platinum wire, and Ag/AgCl (3.0 M KCl)
as working, counter, and reference electrodes, respectively. FT-IR
(KBr) spectra were recorded on Perkin Elmer (model-L1600300
Spectrum TWO LITA), Llantrisant, UK. Surface morphologies of
coatings were studied using scanning electron microscope (SEM)
[JEOL, JSM model-840A (Netherlands)] and atomic force micro-
scope (AFM) [using a NT-MDT microscope, NT-MDT Co. (Russia), in
the semi-contact mode]. A spin coater (ACE → 200, Dong Ah Tech,
Seoul, South Korea) was used for the electrode modification. All
experiments were carried out at 25 ± 1 ◦C.
2.3. Synthesis of AuNPs and functionalized MWCNTs
AuNPs were prepared following the known recipe [24]. In short,
2.5 mL of 1% tri-sodium citrate was added to 100.0 mL of boiling
0.01% HAuCl4 solution. The prepared AuNPs were stored in dark and
refrigerated at approximately −4 ◦C. For evidence, AuNPs showed
a characteristic absorption at ␭max of about 520 nm.

3. B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639 633
Scheme 1. Schematic development of Dual imprinted MIP-AuNPs@PGE.
MWCNTs were functionalized as MWCNTs-COOH following the
known procedure [25]. For this, MWCNTs (0.5 g) were oxidized
with 60 mL of concentrated nitric acid solution at 100 ◦C for 12 h.
After cooling to room temperature, the solid MWCNTs-COOH were
filtered out, washed, and finally dried in vaccum.
2.4. Synthesis of monomer
Monomeric precursor, N-acryloyl pyrrolidine-2,5-dione
(NAPD), was synthesized, following a known method [26], by
reacting succinimide (25 mmol) and acryloyl chloride (25 mmol) at
0 ◦C in THF, in the presence of TEA (25 mmol). The reaction mixture
was maintained at 0 ◦C with stirring for 3 h. After evaporation of
THF, a crude product was obtained. This was generously washed
with water to remove the triethylamine-acid salt and recrystallized
with ethanol.
2.5. Fabrication of MIP with dispersed MWCNTs-COOH on AuNPs
decorated PGE (MIP-AuNPs@PGE)
The fabrication protocol of MIP-AuNPs@PGE sensor is shown
in Scheme 1. PGE was first dipped overnight in the AuNPs solu-
tion. AuNPs were physically adsorbed on the graphite surface
of PGE [27]. The AuNPs@PGE so obtained was dried under the
flow of nitrogen. For the preparation of MIP-AuNPs@PGE, a pre-
polymer mixture was prepared which contained a monomer
(NAPD, 0.4 mmol, 1.0 mL DMSO), d- and l-Asp (0.1 mmol each
dissolved in 1.0 mL DMSO), an initiator (AIBN, 0.003 g), and a cross-
linker (EGDMA, 2.0 mmol). To this mixture, an optimized amount
(20 ␮L) of functionalized MWCNTs (0.005 g MWCNTs-COOH sus-
pended in 800 ␮L DMSO) was added. The whole content was purged
with N2 gas for 10 min, and 15.0 ␮L of this was spin coated on to
the surface of AuNPs decorated PGE at 2600 rpm for 30 s. Herein,
the dispersed MWCNTs-COOH may help for the firm adherence
of MIP-film onto PGE via aromatic ␲-␲ interactions between the
carbon nanotubes and the graphite layers [28–30]. This modified
electrode is subjected to the free radical polymerization at 70 ◦C
for 4 h. Template molecules were finally retrieved from the so pro-
duced MIP-adduct, by immersing the modified electrode into 0.1 M
NaOH plus 0.1 M phosphate buffer (1:2, v/v) mixture for 1 h. The
template removal could be easier from the surface imprinted sites
as obtained by the ‘surface grafting from approach’. The complete
removal of all template molecules was ensured by the gradual
increase of probe response until a maximum is attained. A non-
imprinted polymer modified electrode (NIP-AuNPs@PGE) was also
made in the identical manner as stated above, but in the absence of
template (d-Asp and l-Asp) molecules in the pre-polymer mixture.
2.6. Voltammetric procedure
For electrochemical measurements, MIP-AuNPs@PGE was
immersed into a cell containing 10.0 mL of 0.01 M phosphate buffer
(pH 3.0), in the presence of potassium ferricyanide (33.0 ␮g mL−1,
50.0 ␮L). Before recording CV and DPASV runs, the probe molecules
were accumulated at the electrode surface in the form of electrical
double layer consisting an array of K+ and [Fe(CN)6]3− [31] at −0.4 V
for 150 s. Here, [Fe(CN)6]3− was instantly reduced at accumula-
tion potential −0.4 V. The reduced form [Fe(CN)6]4− was scanned
for CV within the potential window −0.6 to +0.6 V in the anodic
stripping mode. Similarly, DPASV runs were recorded from −0.6 to
+0.4 V at a scan rate of 10 mV s−1 applying pulse amplitude (25 mV),
pulse time (50 ms) and step potential (5 mV). Note that the modified
electrode was found not to be responsive, when it was anodically
charged which restricted the formation of an electrical double layer.

4. 634 B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639
After water-washings, molecular cavities remained occluded with
ferricyanide probe solution that identically responded to initial cur-
rent for the oxidative stripping, [Fe (CN)6]4− → [Fe (CN)6]3−. At this
stage, the dual imprinted electrode was exclusively saturated with
d-Asp until a constant DPASV current (I) was attained, without any
further decrease of probe response. Now, cavities specific to l-Asp
were set free to recapture test analyte and simultaneously release of
the commensurate amount (proportional to l-Asp concentration)
of probe responding decreased DPASV current (I ). Accordingly, the
difference in probe oxidation current ( I = I − I ) versus l-Asp con-
centration profile was obtained. Finally, the modified electrode was
regenerated by retrieving both templates for the next use for d-Asp
estimation. This was carried out following the similar manner as
stated above for l-Asp. For this, molecular cavities for l-Asp were
saturated and d-Asp was evaluated on the same electrode.
Since dissolved oxygen present in the cell did not affect the cur-
rent response, any deaeration of the cell content was not necessary.
The limit of detection (LOD) was calculated as three times the stan-
dard deviation for the blank measurement in the absence of target
analyte divided by the slope of the calibration plot.
3. Results and discussion
3.1. Polymer characteristic
Traditional MIP@PGE did not respond satisfactorily for ferri-
cyanide probe (80 ng mL−1) owing to the insulating nature of acrylic
MIP-film (Fig. 1, curve b). However, the corresponding MWCNTs-
COOH dispersed MIP@PGE revealed 1.3 times higher current (curve
f) as compared to MIP@PGE. The MIP-AuNPs@PGE revealed much
higher current with better electronic transmission (curve g) from
the surface imprinted binding sites to the electrode, even in the
absence of CNTs. The electron transport was drastically improved
when MIP used was duly dispersed with MWCNTs-COOH in the
film texture and modified over AuNPs@PGE surface (curve h). As
a matter of fact, this current height (curve h) was realized just
twice than the MIP film (without MWCNTs-COOH) (curve g) and
thrice than traditional MIP (curve b). This showed that cumu-
lative effect of AuNPs and MWCNTs-COOH imparted significant
electro-conductivity to the MIP film with the channelized electron
transport from the recognition sites to the electrode. Furthermore,
MIP-AuNPs film (with dispersed MWCNTs-COOH) had shown the
electrocatalytic property to some extent to decrease ferrocyanide
oxidation overpotential at −0.18 V vs Ag/AgCl (curve h) as com-
pared to bare PGE (curve c) and AuNPs@PGE (curve e). During
the fabrication of MIP membrane at AuNPs@PGE, the carboxylic
acid groups at the CNT entrance provide an electrostatic ‘gate-
keeper’ effect on ionic transport providing an exciting opportunity
to dramatically enhance the mass diffusion through CNT core. Also,
being electronically conductive, MWCNTs-COOH may localize elec-
tric field at CNT tips to perform electrochemical transformation
[19]. On the other hand, AuNPs in between the porous PGE and the
redox sites of the MIP membrane might serve as “nanomediators”
or “electronic bridges” to trap charge and fortify the channelized
electron transport [32]. The AuNPs electronic bridges help estab-
lishing a fast mediated electron transfer between the redox sites of
the immobilized MIP film and the electrode. Note that the direct
electron transfer between the MIP (with MWCNTs-COOH) and PGE
had demonstrated a restricted probe current (curve f), without
any reduction of overpotential. This is because of the fact that the
direct electron transfer requires a short distance (less than 15–20 Å)
between the redox centre of the MIP film and the electrode surface
[33]. This indicated that AuNPs had exclusive role in this study to
induce electrocatalytic behaviour to the MIP film.
In support of improved electrode kinetics of MIP (with
MWCNTs)/AuNPs@PGE as discussed above, we have calculated
electron-transfer rate constants (k) for the redox process with
probe solution (80 ng mL−1) at bare/modified PGEs, with the help
of following Laviron equation [34], implicating the CV run recorded
at a scan rate of 0.1 V s−1 [Fig. 1 (inset)]:
logk = ˛ log (1 − ˛) + (1 − ˛) log˛ − log
RT
nFv
− ˛ (1 − ˛) nF Ep/2.3RT (1)
where ␣ is the electron-transfer coefficient, F is the Faraday
constant, the scan rate (Vs−1), R the gas constant, T the tem-
perature, and n is the number of electron transfer. For n = 1, the
␣ value could be obtained from the slope (2.303RT/(1 − ␣)nF)
of Ep vs. log plot. The effect of scan rate on CV runs
is shown in Fig. 1 (inset), exclusively for MIP(with MWC-
NTs)/AuNPs@PGE. We have also undertaken the similar study
with other bare/modified PGEs (Fig. not shown). From the cor-
responding Ep vs. log plots, the estimated values of ␣ and k
are: 0.72 and 0.44 × 10−2 (MIP@PGE), 0.73 and 0.48 × 10−2 (bare
PGE), 0.72 and 0.88 × 10−2 (MWCNTs@PGE), 0.70 and 1.24 × 10−2
(AuNPs@PGE), 0.72 and 2.20 × 10−2 (MIP(withMWCNTs)@PGE),
0.75 and 3.24 × 10−2 MIP(without MWCNTs)/AuNPs @PGE, 0.78
and 9.24 × 10−2 s−1 (MIP(with MWCNTs)/AuNPs @PGE). The
fractional ␣ value indicates the quasi-reversible behaviour of ferri-
cyanide probe on each electrode studied. The deviance from ideal
reversibility of probe may be attributed to the difficulty in stripping
of reduced ferricyanide, under the influence of electrical double
layer formed at Eacc = −0.4 V. Nevertheless, the relatively high k
value, which supports the improved electron-transfer kinetics of
the proposed sensor, is due to the cumulative contribution of MWC-
NTs and AuNPs toward augmenting the electron transport.
For developing imprinted network, different template-
template-monomer molar ratios (1:1:1, 1:1:2, 1:1:3, 1:1:4)
were attempted to explore an optimum stoichiometry of the
MIP-adduct complex. The maximum development of DPASV
diminishing current ( I) of both the analytes was obtained when
template-template-monomer ratio of 1:1:2 was used for the poly-
merization at 70 ◦C for 4 h. Insofar as the cross-linker optimization
is concerned, any amount of cross-linker more than 2.0 mmol
revealed a decrease in the I owing to excessive cross-linking
to block the analyte diffusitivity into respective molecular cavi-
ties. Templates from MIP-adduct were retrieved by 0.1 M NaOH
plus 0.1 M phosphate buffer (v/v 1:2), in a sufficient duration of
60 min, under dynamic condition [For details on stoichiometry of
MIP-adduct and optimization of polymerization conditions, vide
Supporting data Section S.1 and Fig. S1].
3.2. Spectral and surface characterization
FT-IR (KBr) spectra (Fig. S2) of monomer (NAPD), template (Asp),
MIP-adduct, and MIP (template-free), were comparatively studied
to propose a tentative binding mechanism between monomer and
template (Scheme 1). Accordingly, MIP possesses two distinct spa-
tial patterns of d-Asp and l-Asp cavities: one in which ␣ − amino
group ( NH3
+) is in the plane and readily accessible for hydrogen
bonding with the host, whereas the other isomer carries amino
group out of the plane and is not available for hydrogen bond-
ing, under steric compression. In the present instance, specificity
of these molecular cavities is not primarily dependent on their
shapes, but also on their respective chemical affinities for the selec-
tive analyte binding, under the impact of phenomenal imprinting
effect. The complexation between the monomer and template(s)
via hydrogen bondings was indicated by the downward shifts of
their respective key bands participating in the adduct formation
[For details on IR characteristics, vide Supporting data Section S.2].
The SEM image of AuNPs@PGE (Fig. 2A) shows somewhat non-
uniform distribution of highly packed and aggregated AuNPs on the

5. B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639 635
Fig. 1. DPASV response of 80 ng mL−1
[Fe(CN)6]3−
(accumulated in the reduced form as [Fe(CN)6]4−
at −0.4 V) at (a) NIP (with MWCNTs)/AuNPs@ PGE, (b) MIP@PGE, (c) bare
PGE, (d) MWCNTs@PGE, (e) AuNPs@PGE, (f) MIP (with MWCNTs) @ PGE, (g) MIP (without MWCNTs)/AuNPs@ PGE, and (h) MIP (with MWCNTs)/AuNPs@ PGE. Inset shows
CV for probe (80 ng mL−1
) recorded in anodic stripping mode at different scan rates (a → e): 10, 50, 100, 200, 500 mV s−1
at MIP (with MWCNTs)/AuNPs@ PGE.
Fig. 2. SEM images: (A) AuNPs, (B) MIP-adduct, (C) MIP, (D) NIP, and (E) side view of MIP- AuNPs films at PGE surfaces.
electrode surface. In contrast, the MIP adduct-AuNPs@PGE (Fig. 2B)
has a relatively compact and rigid structure with clearly visible
AuNPs aggregation within the polymer matrix; MWCNTs are non-
visible in this compact film. Interestingly, upon templates retrieval,
the MIP-AuNPs@PGE surface revealed pores of different depths and
apertures with dispersed MWCNTs in the nanofilm (Fig. 2C), in
contrast to the corresponding NIP-based electrode surface having
almost no pores (Fig. 2D). Fig. 2E displays the side view of MIP-
AuNPs@PGE which suggests the film thickness to be about 95 nm.
Surface morphologies were further supported from AFM (three-
dimensional) images, recorded under semi-contact mode, for MIP
adduct-AuNPs@PGE (Fig. S3A) and MIP-AuNPs@PGE (Fig. S3B). This
also revealed MIP coatings on the electrode surface with thickness
of 94.5 nm [For details on AFM morphology, vide Supporting data
Section S.3].

6. 636 B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639
3.3. Electrochemical studies
First and foremost, all the operating conditions of electro-
chemical analysis were optimized in aqueous conditions using
ferricyanide with MIP-AuNPs@PGE. Accordingly, the accumula-
tion potential (Eacc), the accumulation time (tacc), and pH of the
phosphate buffer, for [Fe(CN)6]3− probe, were obtained as −0.4 V
(versus saturated Ag/AgCl), 150 s, and 3.0, respectively (Fig. S4).
Herein, both potassium and ferricyanide ions occupy and fill up
the imprinted cavities of d-Asp and l-Asp in an open circuit. The
probe gets effectively diffused to the electrode surface, under the
accumulation potential effect at −0.4 V, and arranged there as an
electrical double layer under the pool of electrostatic interactions.
At this potential, [Fe(CN)6]4−, the reduced form of probe, was anodi-
cally oxidized to respond DPASV signal in phosphate buffer solution
(pH 3). This practice was always carried out in the beginning to
record the initial signal of ferricyanide probe that was unaltered,
even after water-washings. This meant cavities remained practi-
cally filled-in with probe solution in the open circuit. At this stage,
L-specific cavities were presaturated with l-Asp (67 ng mL−1) till
no reduction of DPASV current occurred and then subjected to the
d-Asp measurement. For this, the electrode is exposed to d-Asp
solution maintained at pH 3.0, (Fig. S4) for 120 s accumulation in an
open circuit. This electrode was immersed into the cell containing
phosphate buffer. The existing probe molecules in d-Asp imprinted
cavities started diffusing to the electrode under potentiostatic con-
dition which subsequently stripped off to give rise to a diminished
oxidation current. This process was continued with the addition
of d-Asp and measured the reduction in current (Fig. 3A) till it
attained an optimum decrease. At this stage, both D-specific and
L-specific cavities are now completely saturated. For the sequen-
tial analysis of l-Asp, the electrode was refreshed by retrieving both
templates and used as such following the above procedure. Accord-
ingly, now d-Asp specific cavities were blocked by saturating with
d-Asp (66 ng mL−1) and then analysis with L-specific cavities was
carried out (Fig. 3B). Herein, the effective surface area is reduced
due to blocking of imprinted sites with the hydrogen bonded d-
Asp (or l-Asp), leading to the decrease in the DPASV (Fig. 3) or
CV response (Fig. S5). In this work, DPASV was preferred to CV for
the quantitative analysis because of its relatively high sensitivity
in sufficient time scale of voltammetric measurement. The I so
measured could be related to the concentration (C) of analyte in
accordance with the regression equations potrayed in Table 1. The
gradually diminished current of probe with the increase of ana-
lyte concentration attained a constant above 67 ng mL−1 for both
the isomers, due to binding sites saturation. The non-imprinted
modified electrode revealed insignificant I response for probe
molecules upon analyte spiking (Fig. 3) which indicated an excel-
lent imprinting phenomenon.
3.4. Interference studies and sensor endurance
Interference studies were performed for both the enantiomers
with some structurally related co-existing interferents which are
normally found in biological fluids. These were: glutamic acid
(Glu), tryptophan (Trp), proline (Pro), cysteine (Cys), glycine (Gly),
asparagine (Asn), glutamine (Gln), phenylalanine (Phe), histidine
(His), malic acid (Mal), and thyroxine (Thy). The present MIP-
AuNPs@PGE was slightly responsive (Fig. 4c), without subjecting
the electrode to water washing treatment, for some of the indi-
vidual interferents. This response could be termed as non-specific
and false-positive. In fact, such non-specific contribution was first
noticed on the corresponding NIP-AuNPs@PGE for some of the
interferents (Fig. 4a). This contribution could easily be mitigated
simply by water washings (n = 2, 0.5 mL) (Fig. 4b). Therefore, the
MIP electrode was also subjected to similar washing treatment as
Fig. 3. (A) DPASV response of ferricyanide probe (168 ng mL−1
) on MIP-AuNPs@PGE
with l-Asp saturated (67 ng mL−1
) cavities: (b-l) spiking with different d-Asp
concentrations: 0.0, 3.89, 9.58, 13.72, 17.29, 23.21, 29.32, 33.25, 40.68, 50.18,
66.23 ng mL−1
; curve ‘a’ represents initial response with ferricyanide probes duly
filled in both D- and L-cavities of MIP-AuNPs@PGE, and curve ‘m’ represents cur-
rent response of ferricyanide probe on NIP-AuNPs@PGE [operating conditions:
Eacc−0.4 V, tacc 150 s, pH 3.0, and scan rate 10 mV s−1
(for ferricyanide probe)]. (B)
DPASV response of ferricyanide probe (168 ng mL−1
) on MIP-AuNPs@PGEwith d-Asp
saturated (67 ng mL−1
) cavities: (b-l) spiking with different l-Asp concentrations 0.0,
3.99, 7.89, 10.79, 19.78, 24.12, 31.67, 35.57, 43.52, 55.78, 66.12 ng mL−1
; curve ‘a’ rep-
resents initial response with ferricyanide probe duly filled in both D- and L-cavities
of MIP-AuNPs@PGE, and curve ‘m’ represents current response of ferricyanide probe
on NIP-AuNPs@PGE [operating conditions same as above].
a safeguard against false-positives. In a parallel work with binary
mixtures of the template and interferent(s) concomitantly present
in clinically relevant concentration ratio, the MIP-AuNPs@PGE
showed an exclusive response for the template in question (Fig. 4d
and e) in the quantitative manner by means of stereochemical
selectivity. There was virtually no cross reactivity between the
target and the interferent(s) i.e, D-specific MIP-AuNPs@PGE could
not respond l-Asp and vice-versa. Any molecule that is smaller
(Pro, Cys, Gly, Gln), larger (Glu, Trp, Thy, Phe, His) and similar
(Mal, Asn) in size than d- and l-Asp could not be detected on
the proposed sensor. This reflects substrate-selective imprinting
effect in the present instance. Although the smaller interferents
may have an equal opportunity to reach the binding sites but they
still mismatch with molecular cavities in terms of chemical affin-
ity. Interferences were also examined in real samples (Fig. not
shown) which revealed similar behaviour as observed in aqueous
sample. Note that, any probe like entities present in the real sam-
ples may affect the voltammetric measurements. However, such
effect was found to be largely obviated under the massive sample
dilution effect, and therefore all results were found to be quantita-
tive (100%) in this study. Imprinting factors (␣ = MIP-AuNPs@PGE/ i
NIP-AuNPs@PGE) for both the templates (d- and l-Asp) were found as

7. B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639 637
Table 1
Sample behaviour.
Sample Analyte/s Dilution factor Regression equation Linear range
(ng mL−1
)
Recoverya
(%) LODb
(ng mL−1
) Endogenous
concentrationc
(ng mL−1
)
Aqueous d-Asp – I
(␮A) = (0.112 ± 0.0003) + (0.006 ± 0.013) C
3.89–66.23 99–102 1.11 –
l-Asp – I
(␮A) = (0.109 ± 0.0004) + (0.005 ± 0.015) C
3.99–66.12 98–101 1.14 –
CSF d-Asp 40 I
(␮A) = (0.106 ± 0.0003) + (0.009 ± 0.011) C
4.08–69.15 99–102 1.16 1.63 × 102
l-Asp 50 I
(␮A) = (0.105 ± 0.0003) + (0.004 ± 0.011) C
4.12–69.27 99–102 1.17 2.06 × 102
Blood serum d-Asp 1393 I
(␮A) = (0.098 ± 0.0002) + (0.007 ± 0.014) C
4.38–69.42 99–101 1.25 5.99 × 103
l-Asp 1393 I
(␮A) = (0.100 ± 0.0002) + (0.006 ± 0.013) C
4.30–69.38 99–101 1.25 6.10 × 103
Pharmaceutical d-Asp 484 × 103
I
(␮A) = (0.100 ± 0.0003) + (0.007 ± 0.012) C
4.19–68.21 99–101 1.18 2.02 × 106
l-Asp 484 × 103
I
(␮A) = (0.108 ± 0.0003) + (0.006 ± 0.010) C
4.14–68.21 99–102 1.16 2.00 × 106
a
% Recovery = (amount of analyte determined/amount of analyte taken) × 100.
b
LOD based on the minimum distinguishable signal for lower concentrations of analyte (S/N = 3, 95% confidence level).
c
Original concentration obtained by multiplying lower quantitation limit with the dilution factor.
Fig. 4. (A) I response of 50 ng mL−1
ferricyanide probe after spiking with
20 ng mL−1
d-Asp on MIP/NIP-AuNPs@PGE (l-Asp saturated) in the presence of inter-
ferent(s): (a) NIP before wash, (b) NIP after wash, (c) MIP with individual interferents,
(d) MIP with d-Asp and interferent(s) (1:1), and (e) MIP with d-Asp and inter-
ferent(s) in clinically relevant concentration (ng mL−1
) ratios: 1:200 (l-Asp) 1:200
(Glu), 1:200 (Trp), 1:2000 (Pro), 1:2000 (Cys), 1:2000 (Gly), 1:200 (Mal), 1:200 (Asn),
1:200 (Gln), 1:200 (Thy), 1:2000 (Phe), and 1:200 (mixture of all interferents, each
200 ng mL−1
) in aqueous solutions. (B) I response of 50 ng mL−1
ferricyanide probe
after spiking with 20 ng mL−1
l-Asp on MIP/NIP-AuNPs@PGE (d-Asp saturated) in
the presence of interferent(s) (a) NIP before wash, (b) NIP after wash, (c) MIP with
individual interferents, (d) MIP with l-Asp and interferent(s) (1:1) and (e) MIP with
l-Asp and interferent(s) in clinically relevant concentration (ng mL−1
) ratios: 1:200
(d-Asp) 1:200 (Glu), 1:200 (Trp), 1:2000 (Pro), 1:2000 (Cys), 1:2000 (Gly), 1:200
(Mal), 1:200 (Asn), 1:200 (Gln), 1:200 (Thy), 1:2000 (Phe), and 1:200 (mixture of all
interferents, each 200 ng mL−1
), in aqueous solutions.
high as 8.91 and 8.18, respectively using MIP-AuNPs@PGE (Tables
S1 and S2). The selectivity coefficient (k) and the relative selectivity
coefficient (k’) of both the enantiomers with respect to interferents
were also calculated (Tables S1 and S2). The results for k’ for both
the analytes showed the selectivity gained by the imprinting pro-
cess. Accordingly, all interferents have small selectivity (k’ < 15.3%)
on the proposed sensor. It is, therefore, recommended to wash the
electrode after analyte recapture to avoid false-positive results.
3.5. Stability and reproducibility of the proposed sensor
Long-term stability of sensor is an important factor, where
electrode fouling can be a matter of serious concern. This is
observed particularly with real samples, despite their extensive
dilutions. Therefore, DPASV measurements with ferricyanide probe
(60 ng mL−1) were carried out for the analytes, d-Asp and l-Asp
(each 10.80 ng mL−1), both in aqueous and real samples (CSF,
serum, pharmaceutics), using a single modified electrode. This elec-
trode was essentially regenerated and used on every alternate day,
over a period of three weeks. During this period, the modified elec-
trode was able to maintain its original behaviour and no apparent
deviation in current ( I) was noticed. This meant the modified
electrode was perfectly rugged, in aqueous and real samples for
the period of three weeks. However, after three weeks, the cur-
rent response was found to be decreased only about 2.68, 2.70,
2.72, and 2.71% to its initial ( I) response in aqueous, CSF, serum,
and pharmaceutics, respectively. Insofar as electrode-to-electrode
variation is concerned, a series of as many as five modified elec-
trodes was prepared in the identical manner and tested for indirect
analysis with probe solution for rebinding of both the analytes
(10.80 ng mL−1) present in aqueous and real samples. All mod-
ified electrodes responded quantitatively (100%) with RSD 0.45,
0.47, 0.48, and 0.46% in aqueous, CSF, serum, and pharmaceutics,
respectively. This shows that the proposed sensor can be prepared
precisely and used reproducibly with excellent recognition ability.
3.6. Analytical validation
The proposed sensor is compared with a known electrode [21]
for d- and l-Asp determination in CSF by means of Student’s t-test
[(tcal 2.13 < ttab 2.77)], confidence level 95%]. Although both elec-
trodes are reproducible, the present sensor could indirectly detect

8. 638 B.B. Prasad et al. / Sensors and Actuators B 240 (2017) 631–639
both enantiomers in the wide concentration range, with quanti-
tation limit as low as 4.08 ng mL−1 requisite to diagnose epilepsy
and lennox syndrome. Under optimized analytical conditions, the
proposed sensor is validated for the selective and sensitive analysis
of d-Asp and l-Asp in real samples (Table 1). CSF contains both the
enantiomers. Insofar as other biological fluids are concerned, serum
contains predominantly l-Asp with some non-detectable traces of
d-Asp whereas pharmaceutical sample contains only l-Asp. For
demonstrating the feasibility of analysis of both the templates,
we have diluted the real samples with water so as to mitigate
the matrix effect and to move the analysis in the detection range.
Quantitative DPASV measurements with probe are shown in the
case of a representative real sample (CSF) in which both enan-
tiomers are found to be concomitantly present (Fig. S6A and B).
DPASV runs with probe were also represented in diluted human
serum samples duly spiked with d-Asp and l-Asp isomers (Fig. S7A
and B). Upon massive dilution, all real samples approximated their
behaviours very close to the aqueous sample. This is evident from
almost equivalent slopes of the respective linear calibration equa-
tions (Table 1) which indicate negligible matrix complications. This
could be the reason for applying similar operating analytical con-
ditions (Eacc, tacc, and pH) in real samples as those utilized with
aqueous sample analysis. In particular, since diluted (1393-fold)
serum approximated its sample behaviour similar as aqueous sam-
ple, blood serum sample analysis could be accomplished at pH 3,
without any effect of acidity and matrix complications.
The proposed MIP sensor was compared with other known sen-
sors for d- and l-Asp determinations (Table S3). Accordingly, the
detection ability of most of the known techniques was inferior
to this work; and moreover, majority of the techniques were not
properly validated for the application in real samples.
4. Conclusion
For the first time, we have contemplated the one-by-one
approach, using ferricyanide probe on a dual imprinted single PGE,
for ultra-trace analysis of D- and L-enantiomers, prevalent together
or alone, in real samples. This work merits special significance
in view of the fact that the enantioselective analysis of d- and
l-Asp is a challenge, because both the isomers have same oxida-
tion potentials. The proposed MIP-AuNPs@PGE detected d-Asp and
l-Asp present together with detection sensitivities 1.16 ng mL−1
(d-Asp) and 1.17 ng mL−1 (l-Asp), particularly in CSF, which can
be useful in diagnosis of chronic diseases (epilepsy and lennox
syndrome) manifested at stringent limits. The sensor is repro-
ducible, rugged, regenerable, and cost-effective showing excellent
imprinting effect (␣ = 8.91). This assures reliable results, without
any cross-reactivity and false-positives, in clinical settings. The pro-
posed indirect method of sequential analysis on a dual imprinted
electrochemical sensor may be considered novel and versatile in
the sense that any pair of simple molecules can be used as target
analytes (templates).
Acknowledgements
Authors thank University grant commission, New Delhi for
granting a research fellowship to one of us (S.J). Instrumental
facilities procured from Banaras Hindu University are also greatly
acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2016.09.031.
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Biographies
Bhim Bali Prasad is currently working as a professor of Analytical Chemistry in
the Banaras Hindu University (BHU), Varanasi, India. He has mentored 25 Ph.D.
students and published 115 research papers in several reputed international and
national Journals. He received his B.Sc. degree in Chemistry in 1972 and M.Sc.
degree in 1974 form BHU. He obtained his Ph.D. from BHU. He is a recipient of
several national and international awards for his research contributions in Analyt-
ical Chemistry and nano-materials. His research interests include environmental
chemistry, chromatography, electroanalysis, and detection principle for chemical
analysis, nano-technology, and development of biomimetic nano sensors using
molecularly imprinted polymers for clinical, pharmaceutical and biological analysis.
Swadha Jaiswal is currently pursuing Ph.D. at Banaras Hindu University (BHU) under
the supervision of Prof. Bhim Bali Prasad. She received her B.Sc. degree in 2011
and M.Sc. degree in 2013 from BHU, Varanasi. She is recipient of UGC Fellowship.
Her research interest lies in the field of chemical sensor development, molecularly
imprinted polymers, and electro-analytical chemistry.
Kislay Singh is currently pursuing Ph.D. at Banaras Hindu University (BHU) under
the supervision of Prof. Bhim Bali Prasad. She received her B.Sc. degree in 2011
and M.Sc. degree in 2013 from BHU. She is recipient of UGC meritorious research
fellowship. Her research interest lies in the field of chemical sensors, molecularly
imprinted polymers, and electro-analytical chemistry.