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Biomimetic -mineralized multifunctional

Cite this: DOI: 10.1039/d1an01934a
nanoflowers for anodic-stripping voltammetric
immunoassay of rehabilitation-related proteins
a,b c
Fan Cai, Dianping Tang, Jun Wang*d and Yao Lin*a

Received 26th October 2021,
Accepted 14th November 2021
DOI: 10.1039/d1an01934a
rsc.li/analyst
C-reactive proteins (CRPs; an acute-phase protein) in patients with initial acute cerebral infarction neuro-
logical rehabilitation prediction have a significant correlation. In this work, a simple and sensitive anodic-
stripping voltammetric (ASV) immunosensing system was innovatively designed for the quantitative
screening of target CRPs using biomimetic-mineralized bifunctional antibody-Cu3(PO4)2 nanoflowers as
molecular tags. In this system, a monoclonal anti-CRP antibody-anchored microtiter plate was utilized to
specifically capture target CRPs from the sample. For detection, a sandwiched immunoreaction mode
was employed with the antibody-Cu3(PO4)2 nanoflowers in the presence of analytes. Subsequent ASV
measurement of copper ions (Cu2+) released under acidic conditions from the bifunctional nanoflowers
was conducted at an in situ prepared mercury film electrode. The introduction of hybrid nanoflowers
greatly increased the loading amount of copper ions on the molecular tag, thereby amplifying the detect-
able signal of electrochemical immunoassay. Meanwhile, factors influencing the analytical properties of
the electrochemical immunoassay were investigated in detail. By combining the high-efficiency nano-
hybrids with signal amplification, the dynamic concentration range of electrochemical immunoassay
spanned from 0.01 ng mL−1 to 100 ng mL−1 toward the target CRP. The limit of detection was calculated
to be 0.0079 ng mL−1 at 3Sblank criterion. Intra- and interassay imprecisions (relative standard deviations:
RSDs) were less than or equal to 6.72%. Good anti-interference ability, long-term storage stability, and
acceptable accuracy for the evaluation of human serum specimens were observed during a series of pro-
cedures to determine the target protein. In addition, the bifunctional nanoflower-based immunosensing
system offers promise for the simple, cost-effective analysis of disease-related proteins.

Introduction activation of the classical complement pathway.2 Upon arrival

C-reactive proteins (CRPs) are acute-phase proteins of hepatic
origin whose levels have positive correlation with poor out-
comes in patients with acute coronary syndrome, regardless of
the etiology (infection, trauma and inflammation).1 One of the
major functions of CRP is to recognize foreign pathogens and
phospholipid components of damaged cells, resulting in the
a analyzed routinely in clinical practice.
Central Laboratory at the Second Affiliated Hospital of Fujian Traditional Chinese
Medical University, Innovation and Transformation Center, Fujian University of
Traditional Chinese Medicine, Fuzhou 350122, Fujian, P.R. China.
E-mail: yaolin@fjtcm.edu.cn, yaolinfjfz@gmail.com
b
College of Life Sciences, Fujian Normal University, Fuzhou 350117, Fujian, P.R.
China
c for the colorimetric lateral flow immunoassay,7 electrochemi-
Key Laboratory for Analytical Science of Food Safety and Biology (MOE & Fujian
Province), Department of Chemistry, Fuzhou University, Fuzhou 350108, P.R. China
d
Department of General Surgery at The Second Affiliated Hospital of Fujian
Traditional Chinese Medical University, Collaborative Innovation Center for
Rehabilitation Technology, Fujian University of Traditional Chinese Medicine,
Fuzhou 350122, Fujian, P.R. China. E-mail: 1095077121@qq.com
to a medical facility, initial peak CRP levels are not associated
with treatment failure and 28-day mortality but when used in
combination with other biomarkers or APACHE II score, CRP
has higher predictability for mortality in sepsis scores.3 The
measurement of serum CRP concentration may be helpful in
patients with CSF findings consistent with meningitis, with a
negative Gram’s stain result such that the physician considers
withholding antimicrobial therapy.4 In this regard, CRP is
perhaps the most important marker of inflammation and is
Recently, different methods and strategies have been uti-
lized for monitoring serous CRP samples,5 such as biorespon-
sive hydrogel-based surface relief diffraction gratings,6 peroxi-
dase-mimicking nanozymes with surface-dispersed Pt atoms
luminescence-incorporated lateral flow immunoassays,8
tandem giant magneto-resistance biosensors,9 and impedi-
metric immunosensors.10 Among these systems, electro-
chemical immunosensing probes have gained gradually

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increasing interests due to their combined advantages of high-
sensitivity electrochemistry and specific antigen–antibody
immunoreactions.11,12 However, a key issue for the development
of high-efficiency electrochemical immunoassays is to achieve a
strongly detectable electrochemical signal because most antigen–
antibody reactions cannot produce an electrical signal. Enzyme
labels and nano labels are widely used for this purpose.13,14
Relative to enzyme labels, the rapidly emerging research field of
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bio-nano labels opens up new possibilities for bioanalytical appli-
cations.15 One major advantage of using nano labels is that one
can control and tailor their properties in a very predictable
manner to meet the needs of specific applications.16
Typically, nano labels involve inorganic nano labels,
organic nano labels, and organic–inorganic hybrid nano
labels.17 For example, Yu et al. used platinum or gold nano-
particle–labeled secondary antibodies for the design of
immunoassays, respectively.18,19 The biomolecules were phys-
ically adsorbed onto the surface of nanoparticles. One basic
disadvantage of using adsorption is the instability of antibody
conjugation during the measurement.20 Moreover, it was sus-
ceptible to the ionic strength of the test solution. In contrast,
Sun et al. synthesized cross-linked urease nanoparticles for the
labeling of secondary antibodies to determine lipocalin-2 via
carbodiimide coupling.21 Unfavorably, one of the problems
commonly associated with covalent conjugation is the
decrease in biomolecular activity upon exposure to reactive
groups under harsh reaction conditions.22,23 Interestingly, the
emergence of organic–inorganic hybrid nanosystems opens a
new horizon of the use of nanomaterial labels.24 At this point,
organic–inorganic hybrid materials enable the integration of
useful organic and inorganic characteristics within a single
molecular-scale composite, e.g., unique electronic and optical
properties for this promising class of materials. Various
Scheme 1 Schematic representation of the biomimetic-mineralized-functionalized nanoflowers for the anodic-stripping voltammetric immuno-
assay of the C-reactive protein (CRP): (A) synthesis process of anti-CRP pAb2 antibody-Cu3(PO4)2 hybrid nanoflowers; (B) immunoreaction procedure
and square-wave anodic-stripping voltammetric (SWASV) measurement on an in situ mercury drop glassy carbon electrode (GCE) (note: mAb1:
rabbit anti-CRP monoclonal capture antibody; pAb2: rabbit anti-CRP polyclonal detection antibody).
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metal–organic frameworks, e.g., metal-polydopamine frame-
works25 and zeolitic imidazolate frameworks,26 have been uti-
lized as nano labels for the immunoassay development.
However, most biomolecules such as antibodies and enzymes
were covalently conjugated or physically adsorbed onto the
metal–organic frameworks. To improve this issue, the motiv-
ation of our work is to explore a new organic–inorganic nano
label for the development of electrochemical immunoassays.
In this work, we prepared a new electrochemical signal-
generation tag based on biomimetic-mineralized bifunctional
antibody-Cu3(PO4)2 nanoflowers for the anodic-stripping vol-
tammetric detection of CRP (Scheme 1). The antibody-
Cu3(PO4)2 nanoflowers were synthesized by a facile and mild
biomimetic-mineralizing process. The synthesized organic–in-
organic nanotags are not only used to capture the target CRP
in the sample, but also used as signal probes for electro-
chemical measurement via the released copper ions from the
nanoflowers under acidic conditions. The use of antibody-
Cu3(PO4)2 nanoflowers is expected to enhance the loading
amount of copper ions, thus amplifying the voltammetric
signal of electrochemical immunoassays with a sandwiched
reaction mode on the capture antibody-coated microtiter plate.
The major objective of this study is to construct an enzyme-
free nano labelling probe with the bioactivity for the detection
of disease-related biomolecules.
Experimental section
Chemicals and reagents
Rabbit monoclonal [EPR22862-10] antibodies to C-reactive
protein (abbreviation: mAb1; application: IHC-P and WB; reac-
tivity: human; conjugate: unconjugated; Cat# no.: ab256492),
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rabbit polyclonal antibodies to C-reactive protein (abbrevi-
ation: pAb2; application: IHC-P and WB; reactivity: human and
rat; conjugate: unconjugated; Cat# no.: ab227507), and a
human CRP ELISA kit (C-reactive protein) (one-wash 90 min
protocol; sensitivity: 1.23 pg mL−1; range: 1.95–8000 pg mL−1;
sample type: cell culture supernatant, Cit plasma, EDTA
plasma, Hep plasma, serum; detection method: fluorescent;
assay type: sandwiched; reacts with: human) were purchased
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from Abcam (Shanghai, China). All high-binding polystyrene
microplates were acquired from Greiner (Product no.: 655061,
Frickenhausen, Germany). All other chemicals including
bovine serum albumin (BSA, refined grade, ≥98.0%),
CuSO4·5H2O and phosphate-buffered saline (PBS) solutions
with different pH values were obtained from Aladdin
(Shanghai, China). All other reagents were of analytical grade
and used without further purification. Distilled water or ultra-
pure water used in this work was provided by a Millipore puri-
fication system at a resistivity of 18.2 MΩ cm (Branstead, USA).
Synthesis of anti-CRP pAb2 antibody-Cu3(PO4)2 hybrid
nanoflowers
The hybrid nanoflowers based on polyclonal anti-CRP anti-
bodies and Cu3(PO3)2 were synthesized via a facile and mild
biomimetic-mineralizing process similar to previous
works.27,28 In detail, rabbit polyclonal anti-CRP antibodies
(20 μL, 5.0 mg mL−1) were first dispersed into PBS (975 μL,
10 mM, pH 7.4). Then, a CuSO4 aqueous solution (5.0 μL,
100 mM) was rapidly dropped into the resulting solution. After
that, the suspension was gently shaken using a shaker at 4 °C
for 72 h. During this process, organic–inorganic doped nano-
structures were formed on the basis of chemical reactions. The
prepared suspension was centrifuged for 15 min with a cen-
trifugal force of 12 000g at 4 °C. The obtained precipitation
(i.e., pAb2-Cu3(PO4)2 nanoflowers) was dispersed in PBS
(10 mM, pH 7.4) at a concentration of ∼5.0 mg mL−1 and
stored at 4 °C for further use.
Coating of mAb1 on microplates and immunoreaction
processes
The mAb1-modified microtiter plates were prepared as follows:
initially, anti-CRP mAb1 antibodies (50 μL, 10 μg mL−1 in
50 mM sodium carbonate buffer, pH 9.6) were added into each
high-binding polystyrene microplate and incubated at 4 °C for
12 h with the covering of an adhesive plastic plate sealing film.
Then, the microplate was washed three times with a washing
buffer (0.05% Tween 20, v/v in PBS, 10 mM, pH 7.4). Following
that, 300 μL of the blocking buffer (1.0% BSA, w/v, in PBS,
10 mM, pH 7.4) was injected into each well and reacted at
37 °C for 60 min with slight shaking on a shaker. The micro-
plates were washed as explained before. Then, 50 μL of
different concentrations of the CRP standard/sample (May be
diluted with PBS, 10 mM, pH 7.4) and 50 μL of the as-prepared
pAb2-Cu3(PO4)2 suspension (5.0 mg mL−1) were added into the
well, and immunoreacted at 37 °C for 50 min under gentle
shaking using a shaker. The resulting microplate was washed
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again and measured using the following electrochemical
system.
Anodic-stripping voltammetric measurement processes
To carry out the anodic-stripping voltammetric measurement,
a 20 μL aliquot of 1.0 M HNO3 was initially added to each well
to dissolve the pAb2-Cu3(PO4)2 nanoflowers for the release of
Cu2+ ions under slight shaking (∼8.0 min). Then, the resulting
acidic solution containing copper ions was transferred into
2.0 mL acetate buffer (0.2 M, pH 5.6) containing 10.0 μg mL−1
mercury ions (from HgII acetate). The square-wave-anodic-
stripping voltammetric (SWASV) measurement was conducted
using an AutoLab electrochemical workstation (μAUTIII.FRA2.
V, Eco Chemie, Netherlands) with a conventional three-elec-
trode system, containing a platinum-wire as the counter elec-
trode, an Ag/AgCl electrode as the reference electrode, and an
in situ formed mercury drop on a glassy carbon electrode as
the working electrode with an effective working area of
3.14 mm2. The stripping process involved the pretreatment
(1.0 min, +0.6 V) and the accumulation (2.0 min, −1.4 V). All
the SWASV measurements were carried out after a 15-second
rest period (without stirring) in an applied potential from −0.2
V to +0.2 V with a potential step of 4.0 mV, a frequency of 25
Hz, and an amplitude of 25 mV. A baseline correction of the
resulting voltammogram was performed using the μAUTIII.
FRA2.V software.
Results and discussion
Characterization of pAb2-Cu3(PO4)2 hybrid nanoflowers
In this system, the anodic-stripping voltammetric immuno-
assay mainly involves the mAb1-coated microplate preparation,
pAb2-Cu3(PO4)2 and electrochemical measurement. Anti-CRP
mAb1 antibodies were attached to the microtiter plates by
physical adsorption between high-binding polystyrene and the
protein. pAb2-Cu3(PO4)2 hybrid nanoflowers were synthesized
by a one-pot method accompanying a typical chemical reac-
tion. The introduction of the target CRP triggers the sand-
wiched immunoreaction between mAb1 antibody and pAb2-
Cu3(PO4)2. Under acidic conditions, the released copper ions
can be determined by an anodic-stripping voltammetric
method with high sensitivity.
To realize our design, a transmission electron microscope
(TEM; FEI Talo F200S; Thermo Scientific FEI, USA) was
employed to characterize the as-synthesized pAb2-Cu3(PO4)2
hybrid nanostructures. As shown in Fig. 1A, the nanoscales
exhibited a flower-like structure with an average size of 115 nm
diameter. Meanwhile, we also observed many grooves on the
surface of these nanomaterials. Such small and ultra-thin
sheet-like structures were conducive to the reaction of biologi-
cal molecules and the dissolution of particles. The elemental
mapping indicated the presence of Cu, P, O and C elements in
the hybrid nanoflowers (Fig. 1B–E). Logically, another puzzling
question arises as to whether anti-CRP pAb2 antibodies were
doped into the nanoflowers. As a control test, pure Cu3(PO4)2
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Fig. 1 (A) SEM image of pAb2-Cu3(PO4)2 hybrid nanoflowers; (B–E) elemental mapping of Cu, P, O and C elements; (F) UV-vis absorption spectra of
(a) anti-CRP pAb2 antibody, (b) Cu3(PO4)2 nanoparticles and (c) pAb2-Cu3(PO4)2 hybrid nanoflowers.
nanostructures were synthesized by the same method in the
absence of anti-CRP pAb2 antibodies. Thereafter, we used UV-
vis absorption spectrometer (Cary 7000, Agilent, USA) to inves-
tigate three components including Cu3(PO4)2, anti-CRP pAb2
antibody and pAb2-Cu3(PO4)2 nanoflowers, respectively
(Fig. 1F). Curve ‘a’ shows UV-vis absorption spectra of anti-
CRP pAb2 antibodies, and an obvious characteristic peak at
278 nm was observed as a result of the antibody proteins. In
contrast, no characteristic peaks were achieved toward
Cu3(PO4)2 nanoscales within the wavelength of 200–500 nm
(curve ‘b’). Significantly, the characteristic peak for anti-CRP
pAb2 antibodies appeared after the formation of pAb2-
Cu3(PO4)2 hybrid nanostructures (curve ‘c’). On the basis of
these comparative results, we could preliminarily judge the
successful synthesis of pAb2-Cu3(PO4)2 nanoflowers.
Electrochemical characteristics of pAb2-Cu3(PO4)2 under
different conditions
As mentioned above, the anodic-stripping voltammetric signal
of the electrochemical immunoassay stemmed from the solu-
tion of copper ions under acidic conditions toward pAb2-
Cu3(PO4)2 nanoflowers. To demonstrate this issue, a pAb2-
Cu3(PO4)2 suspension before and after the reaction with 1.0 M
HNO3 was prepared in 2.0 mL acetate buffer (0.2 M, pH 5.6)
containing 10.0 μg mL−1 mercury ions using an in situ formed
mercury-drop glassy carbon electrode (Fig. 2A). A very weak
anodic-stripping voltammetric peak was obtained within the
applied potentials for pAb2-Cu3(PO4)2 nanoflowers in the
absence of 1.0 M HNO3 (curve ‘a’), which mainly derived from
the dissolution of Cu3(PO4)2 through an acetate buffer solu-
tion. Favourably, a strong anodic-stripping voltammetric peak
appeared after the reaction of pAb2-Cu3(PO4)2 with 1.0 M
HNO3 (curve ‘b’), which originated from the water-soluble
copper ions. In this case, we also utilized TEM to characterize
the resulting solution, and no particles or nanoflowers were
found in the HNO3-treated solution (data not shown). These
results indicated that the strong anodic-stripping voltammetric
peak appeared due to the copper ion solution, but not pAb2-
Cu3(PO4)2 nanoflowers.
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Fig. 2 (a and b) SWASV diagrams of the various components in 2.0 mL
acetate buffer (0.2 M, pH 5.6) containing 10.0 μg mL−1 mercury ions
(from HgII acetate) toward pAb2-Cu3(PO4)2 (a) before and (b) after the
reaction with 1.0 M HNO3. (c and d) SWASV responses of the developed
voltammetric immunoassay in the (c) presence and (d) absence of the
target CRP (1.0 ng mL−1 used in this case).
Next, the as-prepared pAb2-Cu3(PO4)2 nanoflowers were
used for the detection of target CRP (1.0 ng mL−1 used as an
example) on the mAb1-coated microplate by coupling with the
square-wave-anodic-stripping voltammetric (SWASV) measure-
ments. As seen from curve ‘c’, the obtained voltammetric peak
current was 708.2 nA toward 1.0 ng mL−1 CRP. Maybe, one
question to be produced was whether the SWASV peak was
from non-specific adsorption of pAb2-Cu3(PO4)2. For compari-
son, the pAb2-Cu3(PO4)2 suspension was directly incubated
with a mAb1-coated microtiter plate (i.e., in the absence of the
target CRP). As indicated from curve ‘d’, the voltammetric
peak current was close to zero, suggesting that the strong vol-
tammetric peak currents were obtained from the specific
antigen–antibody reaction between the target CRP and pAb2-
Cu3(PO4)2/mAb2. Therefore, pAb2-Cu3(PO4)2 could be used pre-
liminarily for CRP detection.
Optimization of experimental conditions
As described above, this optimization was carried out through
the antigen–antibody reaction, followed by the anodic-strip-
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ping voltammetric measurement. Generally, the formation of

the immune complexes needs to be carried out within a
certain period of time through the hydrogen bond. In this
work, a monoclonal anti-CRP antibody (mAb1) and a polyclo-
nal anti-CRP antibody ( pAb2) reacted with the target CRP in
the same solution. Fig. 3A represents the effect of immunor-
eaction time on the SWASV peak current of electrochemical
immunoassay using 1.0 ng mL−1 CRP as an example. The vol-
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tammetric currents increased with the increase in immunor-
eaction time and then reached a plateau after 50 min, indicat-
ing that the antigen–antibody reaction reached a dynamic
equilibrium. To save the assay time, 50 min was chosen for the
formation of immunocomplexes.
Another factor influencing the anodic-stripping voltam-
metric current of the electrochemical immunoassay depends
on the amount of the dissolved copper ions under acidic con-
ditions. In this regard, the chemical reaction time between
Cu3(PO4)2 and HNO3 was crucial for the nanoflower dis-
solution. As shown in Fig. 3B, the optimum peak currents were
achieved after 8.0 min, suggesting that the reaction of
Cu3(PO4)2 nanoflowers with HNO3 was relatively slow. To
achieve an adequate signal, 8.0 min was selected for the dis-
solution of Cu3(PO4)2 nanoflowers to obtain the copper ion
solution.
Calibration plots of the electrochemical immunoassay toward
CRP standards
Using the as-prepared pAb2-Cu3(PO4)2 labels, target CRP stan-
dards of different concentrations were determined on the
mAb1-coated microplates with a sandwiched immunoreaction
mode (Scheme 1). The final signal was acquired on the basis
of square-wave anodic-stripping voltammetric measurement
on an in situ formed mercury drop electrode. Fig. 4A shows the
SWASV diagrams of the electrochemical immunoassay toward
different concentrations of CRP standards within the applied
potentials from −200 mV to +200 mV. The overall trend was
that the peak currents increased with the increase in CRP
levels. As seen from Fig. 4B, however, the peak current tended
to be flat (i.e., no obvious changes) within the range of low-
level or high-level CRP standards. Significantly, a good linear
relationship between the decimal logarithm of CRP concen-
trations and SWASV peak currents could be achieved from 10
Fig. 4 (A) SWASV responses of the pAb2-Cu3(PO4)2-based immune
sensing system toward CRP standards with different concentrations, and
(B) the corresponding calibration plots.
pg mL−1 to 100 ng mL−1. The linear regression equation was
fitted to y (nA) = 311.2 × log C[CRP] (ng mL−1) + 709.8 (r =
0.9932, n = 5). The limitation of detection (LOD) was calculated
for 7.9 pg mL−1 CRP at a signal-to-noise ratio of 3. Moreover,
the analytical properties including the linear range and LOD
were acceptable relative to previously reported CRP analytical
methods (Table 1).
Repeatability, specificity and stability of our system
The repeatability of electrochemical immunoassay mainly
involved mAb1-coated microplates and the as-prepared pAb2-
Cu3(PO4)2 nano labels. In this system, mAb1-coated micro-
plates and pAb2-Cu3(PO4)2 could not be repeatedly used for the
detection of target CRP (i.e., disposable) since the hydrogen
bond between the antigen and the antibody is not repeatable.
Based on this consideration, the immunosensing probes with
the different batches were studied for the repeatability. As
shown in Table 2, the maximum relative standard deviations
(RSDs) were 3.04% (n = 4) and 6.72% (n = 4) for the same-
batch (intraassay) and various-batch (interassay) immuno-
sensing probes, respectively, indicating good reproducibility.
The selective detection of the pAb2-Cu3(PO4)2-based
immunosensing system toward CRP is very important for the
newly developed analytical method.37 To monitor this concern,
Table 1 Comparison of analytical properties between the voltammetric
immunoassay and other CRP analytical methods on the linear range and
LOD (unit: ng mL−1)

Method Linear range LOD Ref.

UV-vis absorption 3.0–100 1.2 29

spectroscopy
Lateral flow immunoassay No 0.015 7
application
Lateral flow immunoassay 50–10 000 1.3 30
Voltammetric immunoassay 1.0–106 0.3 31
Electrochemiluminescence 0.01–1000 0.0046 8
Impedimetric immunoassay No 1.2 32
application
Magnetoresistance biosensor 3.0–350 000 1.0 9
Surface acoustic wave sensor 10–100 4.0 33
Fig. 3 Effects of (A) immunoreaction time on the antigen–antibody Colorimetric immunoassay 10–180 000 10 34
reaction and (B) dissolution time of Cu3(PO4)2 with HNO2 on the SWASV Lateral flow immunoassay 100–5000 1.0 35
peak current of the developed immunosensing platform (1.0 ng mL−1 Electrochemiluniescence 0.05–6.25 0.011 36
CRP was used in this case). ASV immunoassay 0.01–100 0.0079 This work

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Table 2 Repeatability of the mAb1-coated microplates and pAb2- 90% of the initial current after 5 and 11 months, respectively,
Cu3(PO4)2 prepared through different batches (conc.: ng mL−1; assay thereby suggesting acceptable stability.
time: nA)
Evaluation of real samples
Conc. 1 2 3 4 RSD (%)
According to local ethics, we collected three human serum
Intrassay 0.1 398.5 394.6 401.2 405.6 1.16
samples including the target CRP from the Department of
10 1119.5 1165.3 1107.3 1178.9 3.04
Interassay 0.1 403.2 389.7 396.5 378.5 2.69 Clinical Laboratory, Fujian Provincial Hospital, China. To
10 1021.3 1132.5 1198.2 1087.4 6.72 investigate the merits of our system, sample no. 2 was diluted
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to different folds with a blank human serum. These samples

other acute-phase reactants [e.g., α-antitrypsin (ATS),
α-antichymotrypsin (ACTS), and ceruloplasmin (CP)] and bio-
markers [e.g., alpha-fetoprotein (AFP) and carcinoembryonic
antigen (CEA)] were measured using our developed electro-
chemical immunoassay (note: the comparison of peak currents
toward these analytes was carried out between the low-level
CRP and high-level non-target). Fig. 5A shows the experimental
results for these analytes. It was found that the strong peak
currents could be acquired in the presence of the target CRP,
and other non-targets gave the very weak signal. Meanwhile,
the voltammetric signal of the mixture including the CRP and
non-target was almost the same as the target CRP alone, thus
exhibiting good specificity.
Certainly, the long-term stability of the prepared immuno-
sensing probes is necessary for CRP analysis. When not in use,
mAb1-coated microplates and pAb2-Cu3(PO4)2 probes were
stored at 4 °C. During the storage period, they were intermit-
tently used to analyze 1.0 ng mL−1 CRP. As seen from Fig. 5B,
the detectable voltammetric signals could maintain 95% and
were assayed using the developed electrochemical immuno-
assay, and the obtained results were compared with those ana-
lysed at the hospital (for the reference). As shown in Table 3,
the RSDs per sample between the two methods were less than
8.99%, indicating the good accuracy for the analysis of real
samples.
Conclusions
In this contribution, we have successfully designed an in situ
amplified anodic-stripping voltammetric immunoassay for the
sensitive detection of C-reactive proteins. This system com-
bined the high-sensitive anodic-stripping voltammetric
method with the specific antigen–antibody immunoreaction.
The immunosensing probes such as mAb1 capture antibody-
coated microplates and pAb2-Cu3(PO4)2 nano labels were
simply prepared or synthesized prior to measurement. The
introduction of strong HNO3 acid was performed after specific
immunoreaction to release the detectable copper ions. The
signal-generation tags with the flower-like Cu3(PO4)2 nano-
structures were favourable for acidic dissolution. In addition,
numerous copper ions could be released from the nano-
flowers, thereby resulting in the ASV signal amplification of
the electrochemical immunoassay.

Compliance with ethical standards

Fig. 5 (A) Selectivity of the pAb2-Cu3(PO4)2-based immune sensing
system, and (B) the storage stability of mAb1-coated microplates and
pAb2-Cu3(PO4)2 probes.
All the experiments were performed in accordance with the
Guidelines of Fujian University of Traditional Chinese
Medicine (China), and approved by the ethics committee at
Fujian University of Traditional Chinese Medicine (China).

Table 3 Measurements for human serum samples before and after dilution using the electrochemical immunoassay and human CRP ELISA kit
(unit: ng mL−1)

Method [mean ± SD (RSD, %), n = 3]

Sample no.a Electrochemistry ELISA kit RSDb (%)

Undiluted 1 78.2 ± 2.1 (2.69) 82.1 ± 1.3 (1.58) 3.44

2 84.6 ± 1.4 (1.65) 79.3 ± 1.9 (2.40) 4.57
3 36.4 ± 1.6(4.40) 38.4 ± 0.9 (2.34) 3.78
Diluted 4 8.16 ± 0.4 (4.90) 8.01 ± 0.7 (8.74) 1.31
5 0.87 ± 0.06 (6.90) 0.93 ± 0.02 (2.15) 4.71
6 0.092 ± 0.007 (7.61) 0.081 ± 0.004 (4.94) 8.99
a
Samples 4–6 were obtained via diluting sample 2 to 10×, 100× and 1000× fold with blank human serum sample. b RSD values were calculated on
the basis of the mean value per sample between two methods.

Analyst This journal is © The Royal Society of Chemistry 2021


Analyst
Informed consent was obtained for any experimentation with
human subjects.
Conflicts of interest
There are no conflicts to declare.
Published on 15 November 2021. Downloaded by N-List College Programme on 12/1/2021 9:32:16 AM.
Acknowledgements
Authors received financial support from the Natural Science
Foundation of Fujian Province (grant no.: 2020J01311402).
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