Microchimica Acta (2019) 186: 485

https://doi.org/10.1007/s00604-019-3524-4

ORIGINAL PAPER

Two colorimetric ampicillin sensing schemes based on the interaction

of aptamers with gold nanoparticles
Omid Heydari Shayesteh 1 & Raouf Ghavami 1

Received: 9 February 2019 / Accepted: 19 May 2019 / Published online: 1 July 2019
# Springer-Verlag GmbH Austria, part of Springer Nature 2019

Abstract
Two kinds of aptasensors for ampicillin (AMP) are described. The assay strategies include the use of gold nanoparticles (AuNPs)
that were modified with (a) a thiolated aptamer (T-Apt), and (b) a non-thiolated polyadenine aptamer (polyA Apt). The AuNPs
and the aptamers were brought to interaction prior to addition of AMP. T-Apt and polyA Apt are adsorbed on the AuNPs by
different mechanisms. The adsorbed aptamer was able to bind the target while preventing non-specific interactions. Remarkably
different optical absorbances (measured at 520 and 680 nm) are produced the absence and presence of AMP. The assay can
selectively recognize AMP even in the presence of species of similar chemical structure. The T-Apt based assay has a linear
response in the 1–600 nM AMP concentration range and a 0.1 nM limit of detection. The respective data for the polyA Apt assay
are 1–400 nM and 0.49 nM.

Keywords Aptasensor . Non-thiolated aptamer . Ampicillin . PolyA aptamer . Gold nanoparticles

Introduction synthesis/modification, easier storage, better reproducibility,

Numerous methods are known for detection of ampicillin
(AMP). These include high-performance liquid chromatogra-
phy [1], liquid chromatography tandem mass spectrometry
[2], colorimetry [3, 4], fluorescent immunoassay [5], electro
chemiluminescence [6] and electrochemistry [7, 8]. These cur-
rent analytical methods consist of some disadvantages such as
being time consuming complicated, require skilled user, ex-
pensive and elaborate instrumentation which limited them as
the on-site assays in practical applications. So it is highly
required that a simple, fast and practical method for the detec-
tion of AMP residues in food products and human body with
favorable selectivity and sensitivity. Aptamers are single-
stranded DNA or RNA molecules able for firmly binding to
their,s specific targets. Aptamers have key advantages over
antibodies including more chemical stability, simpler
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00604-019-3524-4) contains supplementary
material, which is available to authorized users.
* Raouf Ghavami
r.ghavami@uok.ac.ir; rghavami2000@yahoo.com
1
Department of Chemistry, Faculty of Science, University of
Kurdistan, P. O. Box 416, Sanandaj, Iran
lower cost, and especially wider range of recognizable targets
from metal ions, small molecules to macromolecules (such as
proteins), and even viruses and cells [9, 10]. These privileges
have made them a powerful and versatile functional molecular
tool for a various usage such as medical applications [11–14],
bio-imaging [13] food inspection [14, 15] hazard detection
[16, 17], biosensors [18–22]. In the field of biosensor devel-
opment, aptamers are especially preferable for detection of
small molecules because they enable unique techniques for
signal generations that they are not applicable with antibodies
[23]. Due to antibiotics like other small molecules can not act
as an antigen independently, high specificity and affinity an-
tibodies are difficult to prepare, therefore use of aptamer is
preferable to antibody in molecular recognition systems. In
aptamer-based biosensors (aptasensors), aptamer is active in
different analytical methods such as optical and electrochem-
ical. Colorimetric method has been used extensively due to its
low cost, simplicity, practicability and particularly observation
of the color change by the naked eyes [24]. Nanomaterials
have revolutionized in molecular nanoprobe tools.
Particularly, the AuNPs possess unique optoelectronic
properties, which can easily be tuned by varying their size,
shape, and the surrounding chemical environment [25].
AuNPs possess a strong SPR band that results from the
spatial length reduction of electronic motion and the
485 Page 2 of 10
coherent oscillation of the electrons. In addition, the SPR
band of AuNPs has strong distance-dependent properties,
whereby the aggregation of AuNPs of appropriate sizes
(diameter > 3.5 nm) can induce interparticle surface plas-
mon coupling, resulting in a visible color change from red
to blue [26]. Thus, AuNPs provide a practical platform for
colorimetric assay of any target analyte.
Typically in design and construction of colorimetric
aptasensor two strategies including free [27, 28] and adsorbed
[29–31] aptamer were used. In the free aptamer strategy, the
aptamer and analyte were mixed together to allow binding
afterwards exposure to the gold nanoparticles (AuNPs).
Interactions between the non-bound targets and AuNPs sur-
face resulted in a number of false positives responses and
therefore lead to reduction of sensitivity of this colorimetric
method. Currently, one of the best methods to conjugate
aptamers on gold nanoparticles surface is using thiol modified
aptamers [32]. However, studying non-thiolated DNA is im-
portant for several reasons [33] but firstly, the cost of typical
synthesized aptamer is 90% less than thiolated counterpart.
Secondly, fundamental understanding of the interaction be-
tween DNA and gold can be further increased. Finally, new
applications may be derived from this hybrid material includ-
ing biosensor development [34–36] control of enzymatic re-
action [37], nanoparticle synthesis [38], and medicine [39].
Ideally, non-thiolated DNA should adopt an upright confor-
mation just like the thiolated counterpart to achieve molecular
recognition [33].
The main purpose of this study is, the comparison of two
methods including adsorbed T-Apt and polyA Apt on AuNPs
surface, on the performance of colorimetric aptasensor to de-
tect AMP. Herein we use two methods to adsorb Apt on
AuNPs surface, in the first method T-Apt conjugated to
AuNPs, while in the second method polyA modification-free
Apt conjugated to AuNPs prior to target addition. The
adsorbed aptamer can bind the analyte while hindering non-
specific interactions.
Experimental section
Chemicals and materials
The sequence of AMP aptamer (ssDNA) is (5′-(SH)-(CH2)6-
G C G G G C G G T T G TATA G C G G - 3 ′ ) , 5 ′ - G C G G
GCGGTTGTATAGCGG(T)15-(A)12–3′ were obtained from
Bioneer Co., Ltd.(Daejeon, south korea). Acetic acid, trisodium
citrate, ethyl acetate, polydiallyldimethylammonium chloride
(PDDA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), Hydrogen tetrachloroaurate(III), dithiothreitol
(DTT), citrate and ampicillin, amoxicillin, penicillin,
benzylpenicillin and lincomycin obtained from Sigma
Aldrich. The ultrapure water which was obtained via a
Microchim Acta (2019) 186: 485
Millipore Milli-Q water purification system (Billerica,
MA, USA) with an electric resistance >18.2 MΩ cm
used in all experiments. The glassware was rinsed with
aqua regia (mixture of nitric acid and hydrochloric acid,
optimally in a molar ratio of 1:3) and rinsed with ultra-
pure water and dried before use.
Apparatus
Colorimetric assays were recorded on BioTek Synergy HTX
Multi-Mode Reader. A Zeiss EM10C microscope was used
for TEM analyses.
Synthesis of AuNPs
AuNPs were synthesized based on the previously published
protocol [40]. The information is available in supporting in-
formation (synthesis of gold nanoparticles, Fig. S1).
Fabrication of thiolated-aptamer conjugated
to AuNPs (T-apt-AuNPs) probe
100 μM aptamer heated at 90 °C for 3 min and then cooled at
4 °C for 1 h. Heating and immediately cooling of aptamer
causes structural flexibility of the aptamer keep for binding
to AMP. The received T-aptamer (5′-thiol-modified aptamer)
to be in a disulfide form [HOCH3(CH2)5S-S-5′-oligo] that
mercaptohexanol group protects it. DTT as a reducing agent
disrupt any disulfide bonds (-S-S-) and reduce them to free SH
groups that they ready to interaction with gold surface and
adsorbed on AuNPs [32]. 10 μL DTT solution (1.0 N) and
50 μL (50 μM) aptamer were mixed using a vortex mixer. To
ensure that all disulfide groups are fully split, the mixture kept
at room temperature for 1.5 h. Via extracting with ethyl acetate
3 times using 50 μL per extraction, unfavorable thiol seg-
ments and excess DTT remove from the thiol-modified
aptamer mixture. After vortexing the mixture the upper layer
is discarded. The lower layer is T-Apt that ready to react di-
rectly to gold nanoparticles. Afterward, 40 μL AuNPs and
10 μL 0.5 μM T-Apt were incubated together for overnight.
The T-Apts were adsorbed on the surface of AuNPs through
firm covalent bond between SH and gold.
Fabrication of polyA aptamer conjugated AuNPs
(polyA apt-AuNPs)
In the first stage, 1–3 μL of poly A-tailed aptamer (100 μM in
5 mM HEPES buffer, pH 7.6) and 250 μL of AuNPs (10 nM)
was mixed together and briefly vortexed. Afterwards, citrate
buffer with a final concentration of 10 mM (pH 3) was added
to the above mixture. The mixture was incubated at room
temperature for 3 min, pH of solution by adding HEPES buff-
er with a final concentration of 30 mM (pH 7.6) returned to
Microchim Acta (2019) 186: 485
neutral. The mixture 5–10 min was incubated. In the final
stage, the polyA aptamer-modified AuNPs was centrifuged
at 15,000 rpm and upper layer was discarded. In order to
remove free polyA aptamer, pellet was washed with
HEPES (5 mM, pH 7.6). To make sure that all the free
polyA aptamer are removed from the solution, this centri-
fugation and washing method were repeated 4–5 times
and the polyA Apt-AuNPs dispersed in HEPES (5 mM,
pH 7.6) for further use [33].
Adsorbed T-apt ampicillin colorimetric assay
In order to AMP analysis, 50 μL of T-Apt conjugated AuNPs
solution was combined to 10 μL of different AMP concentra-
tions (0–600 nM). After 1 h, PDDA to final concentration of
20 nM was added to the assay solution, and after 5 min the
UV-Vis measurements (400–800 nm).
Adsorbed polyA apt colorimetric assay
In order to AMP analysis, 50 μL of polyA Apt conjugated
AuNPs solution was combined to 10 μL of different AMP
concentrations (0–600 nM). After 1 h, PDDA to final concen-
tration of 20 nM was added to the assay solution, and after
5 min the UV-Vis measurements (400–800 nm).
Results and discussion
Ampicillin (AMP) assay design
The basis of the interaction between aptamer and its analyte
molecule attributed to three important factors including repul-
sive or attractive electrostatic force, hydrogen bonding,
noncovalent aromatic-aromatic interaction (aromatic stack-
ing) and Van der Waals forces [41]. In the attendance of the
analyte, aptamer is folded into its 3D conformation to quite
interact with its target (analyte). Interaction between 3-
dimensional of AMP aptamer and chemical structure of am-
picillin is shown in Fig. 1(a). Beta-lactam antibiotics such as
ampicillin have a beta-lactam ring in their structure. A primary
amine functional group and a benzene ring in the side chain
(circled) of ampicillin molecular structure involved in reaction
between ampicillin and secondary structure of aptamer. Song
et al. have shown that ampicillin aptamer has a lot of affinity to
this side chain with high specificity [42]. According to their
report, “-GGT(T)-” in the loop region of aptamer as well as “-
GC-” base pairing (circled) at the joint of a loop and stem is
responsible for the binding of ampicillin. The strategy for the
colorimetric detection of AMP for both mentioned design (T-
Apt and polyA Apt) is illustrated in Fig. 1. As shown in Fig. 1
(b), in the absence of AMP, T-apt is adsorbed on the surface of
AuNPs by a strong covalent bond between SH and gold [32].
Page 3 of 10 485
Similarly, non-thiolated DNA (polyA Apt) can also be at-
tached through amine groups of DNA base adsorption [43,
44]. PolyA Apt is adsorbed on the surface of AuNPs by spe-
cific noncovalent nucleotide adsorption. A segment of
polyadenine (polyA) can be used to attain a dense DNA at-
tachment. As regards adenine is adsorbed with a higher affin-
ity than thymine, a straight method is to make one end of
polyA aptamer. The thymine chain is used as a spacer to keep
aptamer configuration which is very essential for the strong
interaction between the aptamer and its target. PolyA block to
serve as an anchor on AuNPs (e.g. a replacement of the thiol
functionality) Fig. 1 (c). So AuNPs are stabilized by the
aptamer (T-Apt or polyA Apt) against PDDA-induced aggre-
gation. However, when AMP exists, it forms the complex with
Apt specifically, which pick up the ssDNA layer from AuNPs
competitively. Then the color of the solution changes from red
to purple due to the AuNPs aggregation, according to AMP
concentration.
Optimization of the parameters
Optimization of the reaction parameters for adsorbed T-apt
colorimetric assay
In order to achieve optimal concentration of PDDA that is
sufficient for aggregate the gold nanoparticles, various
PDDA concentrations including 0, 3, 5, 10, 15, 20, 30 and
40 nM are incubated with AuNPs (10 nM) for 15 min. Result
of optimization experiments showed that 20 nM PDDA is
sufficient concentration to aggregate the AuNPs and change
the red color of the gold nanoparticles to blue (Fig. S2).
Therefore 20 nM PDDA applied in this colorimetric assay.
Afterward, the required amount of T-Apt which should be
adsorbed on the AuNPs surface and prevent aggregation of
optimized gold nanoparticles. This optimum concentration of
T-Apt retains the AuNPs red in color against PDDA (20 nM).
For optimization of T-Apt concentration, different concentra-
tions of T-Apt including 0.05, 0.1, 0.25, 0.5, 1, 2 and 3 μM,
were added to a fix concentration of AuNPs (10 nM) in ultra-
pure water. After 20 min, PDDA (20 nM) was added to the
solution, and after incubation for 5 min UV-Vis spectrum was
recorded. Fig. S3 clearly shows that, 0.5 μM T-Apt via attrac-
tive electrostatic force hybridize with PDDA and hinder the
aggregation of gold nanoparticles accordingly retain the red
color of the AuNPs (dispersed state). Optimization of reaction
conditions causes set up a colorimetric aptasensor for quanti-
tative determination of AMP with high sensitivity.
Optimization of the reaction conditions for adsorbed polyA
apt colorimetric assay
The required amount of polyA Apt which should be adsorbed
on the AuNPs surface and hinder aggregation of gold
485 Page 4 of 10 Microchim Acta (2019) 186: 485

Fig. 1 Schematic illustration of a

colorimetric aptasensor based on
gold nanoparticles for the
detection of ampicillin. The
chemical structure of ampicillin
with the secondary structure of
ssDNA ampicillin aptamer shown
in Fig. 1(a). The aptamer
sequence responsible for the
binding of ampicillin was also
reported to be “-GGT(T)-” in the
loop region as well as “-GC-”
base pairing (circled) at the joint
of a loop and stem. In the absence
of AMP, T-apt is absorbed on the
surface of AuNPs by a strong co-
valent bond between SH and gold
Fig. 1(b). In the presence of AMP,
the aptamer interacts with the
AMP and free PDDA aggregate
the AuNPs. Similarly, in the ab-
sence of AMP, PolyA Apt is
absorbed on the surface of AuNPs
by specific noncovalent nucleo-
tide adsorption Fig. 1(c). In the
presence of AMP, the aptamer in-
teracts with the AMP and free
PDDA aggregate the AuNPs

nanoparticles optimized. This optimum concentration of
polyA Apt retains the AuNPs red in color against PDDA
(20 nM). For optimization of polyA Apt concentration, differ-
ent concentrations of polyA Apt including 0.05, 0.1, 0.5, 1,
1.5, 2 and 2.5 μM, were added to a fix concentration of
AuNPs (10 nM) in ultrapure water. After 20 min, PDDA
(20 nM) was added to the solution, and after incubation for
5 min UV-Vis spectrum was recorded. Fig. S4 clearly shows
that, 1 μM polyA Apt via attractive electrostatic force hybrid-
ize with PDDA and hinder the aggregation of gold

Fig. 2 Sensitivity of the aptasensor for AMP detection using adsorbed T- A680/A520 were fitted to a linear plot with a correlation coefficient of
Apt design. a Absorbance spectra of AuNPs in the presence of increasing 0.991. The inset shows the corresponding visual color changes of the
AMP concentrations. b Calibration plot of the aptasensor. The values of assay solutions
Microchim Acta (2019) 186: 485
Fig. 3 Sensitivity of the aptasensor for AMP detection using adsorbed
PolyA Apt design. a Absorbance spectra of AuNPs in the presence of
increasing AMP concentrations. b Calibration plot of the aptasensor. The
nanoparticles accordingly retain the red color of the AuNPs
(dispersed state).
Spectrophotometric quantitative determination
of ampicillin
In citrate-capped gold nanoparticles, electrosteric stabilization
is the major factor for AuNPs to remain dispersed (red color)
in aqueous solution. Citrate anions, are located between the
gold nanoparticles, causes their electrostatic stability. But in
the presence of the positively charged cationic polymer
(PDDA) and electrostatic attractive between it and citrate,
the electrostatic balance in the solution disappears and leads
to the aggregation of gold nanoparticles (blue color). Herein,
the cationic polymer PDDA was employed to aggregate
AuNPs. In this work, two kinds of ssDNA-functionalized
AuNPs, T-Apt and polyA Apt, were synthesized through
self-assembly. They are attached to AuNPs from 5′ terminal
and 3′ terminal sequences, respectively. When AMP is absent,
T-Apt or polyA Apt that adsorbed on the surface of AuNPs
due to negatively charged phosphate backbone of aptamer
forms duplex with cationic PDDA. Hence, Due to the lack
of enough PDDA, the aggregation of AuNPs is hindered.
Nevertheless After adding AMP, T-Apt or polyA Apt
Table 1 Comparison with
currently available methods for Method of detection
the detection of ampicillin
Colorimetry
Fluorescent Immunoassay
Electrochemistry
Aptasensor detection
Aptasensor detection
Aptasensor detection
Aptasensor detection
Page 5 of 10 485
values of A680/A520 were fitted to a linear plot with a correlation
coefficient of 0.981. The inset shows the corresponding visual color
changes of the assay solutions
transformed to the 3-dimensional structure and because of
higher affinity to AMP than PDDA, forms complex with
AMP. Therefore, the PDD released and caused to the aggre-
gation of gold nanoparticles. Accordingly, remarkable change
occurs in the color of gold nanoparticles from red (λmax =
520 nm) to blue (λmax = 680 nm) that recognizable with the
naked eyes. This color change is related to the concentration
of PDDA which is straightly depends on the concentration of
AMP. Hence, the present two methodologies including
adsorbed T-Apt and polyA Apt on AuNPs can be employed
for detecting of ampicillin colorimetrically.
UV–Vis spectroscopy, TEM and visual detection used for
evaluate the sensitivity of this aptasensor. Different concentra-
tions of AMP from 0 to 600 nM were added into the assay
solutions and then the UV-Vis spectrum from 400 to 800 nm
was recorded. Figure 2 (adsorbed T-Apt method) and Fig. 3
(adsorbed polyA Apt method) clearly show that during in-
creasing concentration of AMP, characteristic peak (λmax =
520 nm) of gold nanoparticles reduction while a new peak
appears (λmax = 680 nm) which indicates the aggregation of
AuNPs. Accordingly, color of assay solution will have an
obvious change from red (dispersed mode), purple to blue
(aggregated mode). For quantitative measurement of AMP,
the variations of absorbance values (A680/520) were plotted
Limit of detection (nM) References
463.60 [3]
6.86 [5]
670 [7]
5.72 (by fluorescence) [42]
28.60 (by colorimetry)
0.28 [47]
0.20 [48]
0.10 (by adsorbed T-Apt method) This work
0.49 (by adsorbed PolyA-Apt method)
485 Page 6 of 10 Microchim Acta (2019) 186: 485

Fig. 4 The transmission electron

microscope (TEM) images of
gold nanoparticles treated with
various substances. a AuNPs, b
AuNPs on the addition of 20 nM
PDDA, c AuNPs after treatment
with 20 nM PDDA and 0.5 μM T-
Apt, d AuNPs after treatment
with 20 nM PDDA and 1 μM
PolyA Apt, e AuNPs after treat-
ment with 0.5 μM T-Apt, 10 nM
AMP and 20 nM PDDA. f AuNPs
after treatment with 1.0 μM
PolyA Apt, 100 nM AMP and
20 nM PDDA. The inset shows
the respective images

and calibration curve was obtained. The A680/520 is favorable
proportionate to the AMP concentrations with a linear plot. In
adsorbed T-Apt method, as demonstrated in Fig. 2(b) the ratio
of absorbance values (A680/520) is fitted to the concentration of
AMP with a linear plot (R = 0.9910). Similarly, in adsorbed
polyA Apt method as demonstrated in Fig. 3(b) the ratio of
absorbance values (A680/520) is fitted to the concentration of
AMP with a linear plot (R = 0.9810). The formula 3α/slope
was used for calculating of detection limit [45, 46], where α
display the standard deviation of the apparatus and slope is
derived from linear calibration plot. The detection limit of the
aptasensor came out to be 0.1 nM and 0.49 nM in adsorbed T-
Apt and adsorbed polyA Apt methods respectively. The pro-
posed methods were linear in the concentration range of 1–
600 nM and 1–400 nM in adsorbed T-Apt and adsorbed polyA
Apt designs, respectively. Also based on visual detection, in
adsorbed T-Apt and polyA Apt strategy, AMP in the concen-
tration of 10 and 50 nM was detected by the naked eyes,
respectively. Compared with the reported methods for ampi-
cillin detection, these proposed bioassays are relatively high
compared to those of a previously reported optical and elec-
trochemical biosensor, which was shown in Table 1. In order
to confirm principle of this colorimetric bioassay, aggregation
behavior of AuNPs was further investigated by analyzing the
Microchim Acta (2019) 186: 485
Fig. 5 Selectivity of the assay for the detection of AMP. a The
absorbance spectra of AuNPs with various antibiotics. The
concentration of all the antibiotics, PDDA and T-aptamer was 20 nM,
samples via TEM. As shown in Fig. 4(a), the gold nanoparti-
cles in their natural state monodispersed and spherical in es-
sence. The addition of an optimized concentrate of PDDA to
assay solution because of its interaction via attractive electro-
static force with citrate anions that are located between gold
nanoparticles, AuNPs aggregated and thereby exhibiting the
solution blue color (Fig. 4(b)). However, T-Apt or polyA Apt
that adsorbed on the surface of AuNPs due to negatively
charged phosphate backbone of aptamer forms duplex with
cationic PDDA. Hence due to the lack of enough PDDA,
AuNPs were maintained in the dispersive mode and the color
of assay solution remained red (Fig. 4(c), (d)). Upon the ad-
dition of AMP to the solution, due to high affinity of aptamer
(T-Apt and PolyA) to AMP, PDDA again becomes free and
Fig. 6 Selectivity of the assay for the detection of AMP. a The
absorbance spectra of AuNPs with various antibiotics. The
concentration of all the antibiotics, PDDA and PolyA Apt was 20 nM,
Page 7 of 10 485
0.5 μM, and 600 nM respectively. b Relative response to the aptasensor
on treatment with different antibiotics. The inset shows the corresponding
images
lead to aggregation of AuNPs by electrostatic interaction into
larger clusters. Gold nanoparticles capped with citrate anion,
maybe electrostatic attraction between cationic PDDA and
citrate anion lead to come closer to AuNPs and aggregated.
As a result of this interaction, color of solution change to blue
(Fig. 4(e), (f)).
Selectivity of the assay
Aptasensor selectivity for AMP detection was also evaluated.
For this, a number several of competing non-target antibiotics
including amoxicillin, penicillin, benzylpenicillin, and linco-
mycin were individually added to the assay solutions, and the
variations of absorbance values (ΔA) were calculated. Results
1 μM, and 200 nM respectively. b Relative response to the aptasensor on
treatment with different antibiotics. The inset shows the corresponding
images
485 Page 8 of 10
Table 2 Determination of AMP by adsorbed T-Apt and adsorbed PolyA-Apt methods in spiked milk samples
Sample Added (nM) Detected (nM)
T-Apt PolyA-Apt
1 10.00 9.82 9.77
2 50.00 50.83 49.12
3 200.00 199.32 205.91
(Fig. 5(a), (b)) and (Fig. 6(a), (b)) showed that changes in
color from red to blue and variations of absorbance values
(ΔA), was observable only for AMP. The biosensor works
specifically only with ampicillin and gives an almost insignif-
icant response to other antibiotics. Moreover, both designs
assay that uses of T-Apt (Fig. 5) and polyA-Apt (Fig. 6) in
construction of aptasensor do not significantly differ in the
specific recognized to ampicillin.
Detection of ampicillin in real samples
The two fabricated aptasensors with two assay strategies in-
cluding adsorbed thiolated aptamer (T-Apt) and adsorbed non-
thiolated polyadenine aptamer (polyA Apt) were evaluated for
examine their ability to determination of AMP in milk sam-
ples. For this purpose, the recoveries of three different con-
centrations of AMP in spiked milk samples were determined
using these aptasensors. Artificially contaminated milk was
prepared by spiking a standard AMP solution into AMP-free
milk. 5 g of AMP-free milk was spiked with the desired con-
centration of AMP, mixed by manual shaking for 2 min and
keep in 4 °C in the refrigerator for 25 min to allow the equil-
ibration of the AMP with the milk matrix. The complex com-
position and the turbidity of milk often restrict direct colori-
metric detection. Thus, pretreatment is usually required to
remove the main interferences and to make the samples trans-
parent. The protocol used for pretreatment of spiked milk
samples is detailed below. Firstly, the pH of samples by added
drop to drop of 20% acetic acid were set to 4.6. Afterwards, in
order to precipitate casein the samples 15 min were incubated
at 45 °C and then for eliminate of proteins and fats centrifu-
gation 20 min at 10,000 rpm. Eventually, after filtered the
samples with a 0.22 mm membrane the pH returned to
neutral [49]. Then via mentioned both methods, the col-
orimetric bioassay accomplish as described above. For
quantitative measurement of AMP in milk samples, the
variations of absorbance values (A680/520) were plotted
and calibration curve was obtained. Like AMP detection
in standard samples, The same linear detection ranges of
1–600 nM and 1–400 nM were obtained in adsorbed T-
Apt and adsorbed polyA Apt assay schemes, respectively.
The two linear regression equations are y = 0.0014x +
Microchim Acta (2019) 186: 485
Recovery (%) RSD (%, n = 3)
T-Apt PolyA-Apt T-Apt PolyA-Apt
98.20 97.70 1.15 0.98
101.66 98.24 0.13 1.29
99.66 102.95 0.26 0.49
0.2775, R 2 = 0.992 and y = 0.0022x + 0.2559, R 2 =
0.981, where y the ratio of the absorbance (A680/520) and
x represents the concentration of AMP (nM), in adsorbed
T-Apt and adsorbed polyA Apt assay schemes, respectively.
As shown in Table 2, the recoveries were in the range of
98.20–101.66% and 97.70–102.95% for adsorbed thiolated
aptamer (T-Apt) and adsorbed non-thiolated polyadenine
aptamer (polyA Apt) assay strategies, respectively. Results
indicate that both developed aptasensors are adequate for the
determination of AMP in food samples.
Conclusions
A colorimetric bioassay based on aptamer-gold nanoparticle
for ampicillin determination has been developed based on the
aggregation of AuNPs controlled by the interactions among
ampicillin, ampicillin aptamer and PDDA, with high selectiv-
ity. In this comparative study, the role of two adsorbed Apt
methods (T-Apt and PolyA Apt) on the AuNPs surface on
performance of colorimetric aptasensor to detect AMP were
investigated. In adsorbed T-Apt ampicillin colorimetric assay
detection of ampicillin was linear in the concentration range
1–600 nM with 0.1 nM as the limit of detection and in
adsorbed polyA Apt ampicillin colorimetric assay was lin-
ear in the concentration range 1–400 nM with 0.49 nM as
the limit of detection. Also based on visual detection, in
adsorbed T-Apt and polyA Apt strategy, AMP in the con-
centration of 10 and 50 nM was detected by the naked
eye, respectively. However in design and construction of
many aptasensors thiolated modified aptamer were used,
the results of our work clearly showed that compared whit
thiolated modified aptamer, polyA unmodified aptamer
can be used in the preparation of aptasensors with com-
parable sensitivity and performance. Therefore, in the
construction of aptasensors, the polyA Apt can be used
as a substitute for the T-Apt to measure a lot of analytes
with satisfactory sensitivity.
Compliance with ethical standards The author(s) declare
that they have no competing interests. The authors alone are responsible
for the content and writing of the paper.
Microchim Acta (2019) 186: 485
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