Current Research in Biotechnology 4 (2022) 58–65

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Current Research in Biotechnology

journal homepage: www.elsevier.com/locate/crbiot

Electrochemical platform for anti-cardiolipin antibody detection in human

syphilitic serum
Elton M.N. Do Egito a,b, Alberto G. Silva-Júnior a,b, Raiza P.S. Lucena a,b, Maria D.L. Oliveira a,b,
César A.S. Andrade a,b,⇑
a
Programa de Pós-Graduação em Inovação Terapêutica, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil
b
Laboratório de Biodispositivos Nanoestruturados, Departamento de Bioquímica, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Syphilis is a sexually transmitted disease with high morbidity and mortality rates. Venereal disease research
Biosensor laboratory (VDRL) and rapid plasma reagin (RPR) testing are the most commonly used syphilis identification
Syphilis methods; however, both have low specificity. The available molecular assays are expensive and time‐
Cardiolipin consuming. Electrochemical biosensors are an innovative approach for detecting multiple target analytes pre-
Cyclic voltammetry
sent in syphilis complex samples. In this work, we used a self‐assembled monolayer (3‐mercaptopropyl)trime
Electrochemical impedance spectroscopy
thoxysilane (MPTS) to immobilize an antigenic colloidal suspension (containing cardiolipin, cholesterol, and
lecithin) as a biorecognition element in a platform for detecting anti‐cardiolipin (anti‐C) antibodies in infected
human serum. The biosensor platform was evaluated through cyclic voltammetry, electrochemical impedance
spectroscopy, and atomic force microscopy. Microscopic evaluation revealed the formation of small
cardiolipin‐anti‐C complexes at the maximum titer of 1:32, thus indicating the serum’s syphilis infection pos-
itivity. Anti‐C antibody detection was evaluated by blocking electron current and oxidation–reduction pro-
cesses at the electrode–electrolyte interface. The proposed sensor yielded a linear response (serum dilutions
ranging from 1:8 to 1:1024 titer) with a regression coefficient of 0.97. We obtained a limit of detection of
1:1024 titer and high selectivity against interfering biomolecules. The developed biosensor may provide a
promising alternative for syphilis diagnosis and follow‐up treatment.

1. Introduction and treponemal assays (Solaimalai et al., 2020). Nontreponemal assays

Sexually transmitted diseases remain a global problem despite
extensive interventions through public health campaigns and medical
advances. According to the World Health Organization, approximately
1 million cases are reported daily worldwide, primarily gonorrhea,
chlamydia, and syphilis (Unemo et al., 2017). More than 20 million
cases of sexually transmitted diseases occur in the United States every
year, and the economic impact of diagnosis and treatment has been
estimated to be approximately US$ 17 billion annually (Barrow
et al., 2020). Syphilis alarmingly infects more than 6 million people
every year, and an estimated 300,000 annual neonatal deaths are asso-
ciated with the disease (Kojima and Klausner, 2018). Treponema pal-
lidum, the causative agent of syphilis, is transmitted through sexual
intercourse, skin contact with lesions, and congenitally.
The traditional methods for syphilis diagnosis are venereal disease
research laboratory (VDRL) testing, rapid plasma reagin (RPR) testing,
are the gold standard. False‐positive results and pro‐zone phenomena
are observed for patients with chronic diseases or those requiring drug
injection devices (Ogden et al., 2019). Moreover, circulating antibod-
ies produced after prior exposure to T. pallidum can also lead to false
positivity in treponemal assays (Park et al., 2019). Molecular assays
(e.g., polymerase chain reaction) are a common alternative to syphilis
diagnosis but are time‐consuming and require expensive equipment/
reagents (Sheng et al., 2010). RPR antigen suspension is composed
of cardiolipin, lecithin, and cholesterol. This suspension is similar to
reagins (antigens released by T. pallidum) and has been widely used
in non‐treponemal tests to detect anti‐cardiolipin antibodies (anti‐C)
(Alhabbab, 2018; Clyne and Jerrard, 2000).
Innovative sensor tools are needed to enable low‐cost, specific, sen-
sitive, and rapid screening. Previously, Kussrow et al. (2010) have
described a versatile approach for assessing antigen–antibody complex
formation by using backscattering interferometry. Surface plasmon

⇑ Corresponding author at: Departamento de Bioquímica, UFPE, 50670-901 Recife, PE, Brazil.
E-mail address: csrandrade@gmail.com (C.A.S. Andrade).

https://doi.org/10.1016/j.crbiot.2022.01.001
Received 21 September 2021; Revised 2 January 2022; Accepted 3 January 2022

2590-2628/© 2022 The Authors. Published by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
E.M.N. Do Egito et al.
resonance has also been used for syphilis antibody detection (Severs
et al., 1993). Recently, an electrochemical DNA scaffold platform has
been developed with T. pallidum N17 antigen (TpN17) as a biorecogni-
tion element for detecting specific T. pallidum antibodies in serum sam-
ples (Ogden et al., 2019). However, TpN17 antigen is a lipoprotein
that is difficult to obtain and has unknown cost, thus hindering
research applications. The antigenic suspension used in RPR and VDRL
tests has similarities to reagin. The antigenic suspension is an afford-
able, easily obtainable reagent that forms immune complexes with
the anti‐C antibodies present in syphilitic serum (Alhabbab, 2018).
Biosensors are a promising tool in diagnostic biomedical fields, because
they provide the advantages of high accuracy, low cost, miniaturization,
and rapid analysis of multiple analytes (Maduraiveeran et al., 2018).
Self‐assembled monolayers have been used to modify electrode surfaces
(Singh et al., 2020). 3‐mercaptopropyltrimethoxysilane (MPTS) has a thiol
group in its structure, which reacts with metal surfaces (Gothe et al.,
2018). In this work, we report the development of an immunosensor to
detect anti‐C antibodies. The sensor platform is based on MPTS monolay-
ers and RPR antigen (recognition element). The proposed sensor is
designed for single use. Cyclic voltammetry (CV) and electrochemical
impedance spectroscopy (EIS) were used to assess the biosensor’s assembly
process and evaluate anti‐C antibody detection. The immune complexes
were evaluated through optical microscopy and atomic force microscopy
(AFM). To our knowledge, this is the first study aiming to develop an
impedimetric biosensor based on RPR antigen and MPTS monolayers to
detect syphilis antibodies in human serum.
2. Experimental
2.1. Materials
Bovine serum albumin (BSA) and MPTS were obtained from Sigma‐
Aldrich (USA). A colloidal suspension composed of cardiolipin, choles-
terol, and lecithin (RPR antigen) was obtained from Diagnostic Wama
(Switzerland). Potassium ferrocyanide (K4[Fe(CN)6]) and potassium
ferricyanide (K3[Fe(CN)6]) were purchased from VETEC (Brazil). The
ultrapure water used throughout the experiments was obtained from
a Milli‐Q plus (Billerica, USA) purification system.
2.2. Blood screening and optical microscopy
Human blood samples (n = 3) were obtained through peripheral
venipuncture (without anticoagulants) in the Clinical Laboratory Gil-
son Cidrim LTDA (Recife, Brazil). The samples were centrifuged at
3000 rpm for 15 min to obtain human serum. VDRL was performed
according to a standard method using a colloidal solution of RPR anti-
gen and Kline plates to titrate the samples. Immune complexes were
visualized under a Nikon Eclipse E200 microscope. An enzyme
immunoassay (CAPTIATM Syphilis (T. Pallidum)‐G) was used for the
detection of IgG antibodies to T. pallidum in serum specimens, accord-
ing to the manufacturer’s instructions (Table S1).
2.3. Electrode surface modification
Before each experiment, a gold disc electrode (ϕ = 2 mm) was pol-
ished with α‐Al2O3 powder (0.05 µm) and washed ultrasonically in
deionized water. The electrode was then incubated for 5 min in MPTS
solution (1 M), thus yielding a nanostructured film. After the physical
adsorption of a colloidal solution of cardiolipin (2 µL), we obtained the
MPTS_RPR biosensor platform. We immobilized BSA (1 mg mL−1) as a
blocking agent on the sensor platform (Wang et al., 2017). Fig. 1
shows a scheme of the biosensor assembly process. We evaluated three
VDRL positive serum samples for syphilis at different titers (1:8, 1:16,
1:64, 1:256, 1:512, and 1:1024) diluted in phosphate buffered saline
(PBS) for 5 min. After each step, the sensor was washed with PBS
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Current Research in Biotechnology 4 (2022) 58–65
(pH 7). The colloidal antigenic suspension formed thin films on the
MPTS monolayer. However, micelles have outstanding structural sta-
bility in solution (Frolov et al., 2011). Multiple interfering analytes
were tested to analyze the selectivity of the sensor platform. The inter-
fering molecules were analytes present in human plasma (glucose, gly-
cine, and citric acid), serum from a healthy baby, and pure anti‐dengue
virus antibodies (anti‐DENV3).
2.4. Electrochemical measurements
Electrochemical characterization of the biosensor assembly process
and analyte identification were performed with an Autolab PGSTAT
128 N potentiostat (Eco Chemie, the Netherlands) interfaced with
NOVA 1.11. A three‐electrode system was used throughout the exper-
iments inside a Faraday cage, and Ag/AgCl (saturated KCl) was used as
the reference electrode. Platinum wire and a bare gold electrode (BGE)
disk (ϕ = 2 mm) were used as the auxiliary and working electrodes,
respectively. All electrochemical studies were performed at 24 °C ± 2
°C with a solution of 10 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) in PBS
(pH 7.4) as a redox probe. EIS spectra were recorded in the frequency
range of 100 mHz to 100 kHz. The amplitude of the applied sine wave
potential was 10 mV. CV was performed with a potential sweep
between + 0.7 and −0.2 V at a scan rate of 50 mV s−1. At least ten
repeated measurements were performed for the sera from three
patients at room temperature (24° C ± 1° C) and inside a Faraday cage.
Sensor‐to‐sensor reproducibility was evaluated according to the coeffi-
cient of variance, and the variation ranged from 6% to 10%.
2.5. Atomic force microscopy
AFM was used to evaluate the morphology of the sensor platform and
its interaction with specific anti‐C antibodies. Data were obtained with
an SPM‐9700 microscope (Shimadzu Corporation, Japan) under ambient
conditions. The AFM measurements used the following parameters:
125 μm length, 30 μm width, 4 μm thick non‐contact/tapping mode–high
resonance frequency silicon probes (Nanoworld, Japan) at a spring con-
stant of 42 N m−1 and a resonance frequency of 300 kHz. All represen-
tative images were chosen from a 30 × 30 μm area, taken at 24 °C (±2°
C) with a scanning area of 5 × 5 µm and a scan rate of 1 Hz line s−1, and
processed in Gwyddion software.
3. Results and discussion
3.1. Topographic analyses
Three‐dimensional AFM images and cross‐sectional analyses of the
electrode surface modifications are shown in Fig. 2 and Fig. S1, respec-
tively. The gold electrode surface topography is shown in Fig. 2a. A
well‐formed MPTS self‐assembled monolayer was chemisorbed on
the electrode surface (Fig. 2b). The modification occurred through a
chemical interaction between the gold electrode surface and the termi-
nal thiol groups. The average roughness of the MPTS film
was ∼ 40 nm. According to Jiang and Cheng (2009), MPTS films have
a well‐oriented compact structure. A maximum height of ∼ 95 nm was
obtained after immobilization of the antigenic system (RPR antigen)
(Fig. 2c). The difference of ∼ 55 nm between the heights of the MPTS
and MPTS‐RPR films corresponded to the approximate size of a micel-
lar antigenic system (Ouanezar et al., 2012).
Fig. 2d and 2e present the biosensor’s topography after exposure to
serum samples from patients infected with Treponema pallidum and
healthy individuals, respectively. Clear increases in the height and
roughness of the sensor system were observed when the antigenic
complex interacted with the infected clinical samples. The presence
of peaks with ∼ 130 nm height demonstrated the ability of the biode-
vice to detect syphilitic infection (Fig. 2d). The interaction of the
E.M.N. Do Egito et al. Current Research in Biotechnology 4 (2022) 58–65

Fig. 1. Schematic representation of the biosensor assembly process.

Fig. 2. 3D AFM images (5 µm × 5 µm) of the gold electrode surface (a), MPTS layer (b), RPR antigen (c), and platform after anti-C antibody interaction (d) and
after uninfected serum sample interaction (e).

biosensor with clinical samples from uninfected patients did not cause
significant morphological alterations (Fig. 2e). The height difference
between the infected and uninfected samples is attributable to the
presence or absence of formation of the antigen–antibody complex
on the sensor surface (Aydın et al., 2018).
3.2. Microscopic characterization of immune complexes
VDRL testing was performed on serum samples to estimate the
immune complexes formed between the cardiolipin present in the RPR
antigen and the anti‐C antibodies. The technical procedure involved mix-
ture of serum (v = 50 µL) and 30 µL of the RPR antigen colloidal solution
on Kline plates under stirring for 3 min (Eaton, 1952). Positive samples
were subjected to serial dilution in PBS. Fig. S2a shows images of the neg-
ative samples for anti‐C antibodies. In that case, no immune complex for-
mation was observed. RPR/anti‐C complexes at titers of 1:32 and 1:8 are
shown in Figs. S2b and S2c. Positive samples were then diluted to various
titers (1:8, 1:16, 1:64, 1:256, 1:512, and 1:1024) and tested with the elec-
trochemical sensor. Higher titers are associated with T. palladium attack
and disruption of host cells (Gao et al., 2018).

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E.M.N. Do Egito et al.
3.3. Electrochemical characterization of the biosensor platform
Variations in the anodic and cathodic peaks corresponded to the block-
ing effect in the electrode/electrolyte interface. Therefore, the sensor
assembly process was evaluated through CV and EIS (Fig. S3). The BGE
showed a reversible cyclic voltammogram with well‐defined redox peaks,
revealing a conductive surface with an anodic peak current (iPA) of 61.0
63 ± 0.581 µA (Fig. S3a). A decrease in the voltammetric peaks was
observed after deposition of the MPTS layer, thus resulting in iPA = 48.
906 ± 1.077 µA (Fig. S3a). The covalent binding of gold‐thiol (Au‐S)
results in a self‐assembled monolayer that blocks electron transfer on
the BGE surface (Hasan and Pandey, 2018).
The MPTS layer immobilized on the gold surface with its rough mor-
phology provides numerous sites for RPR antigen chemisorption, through
the interaction of –OCH3 and the micelles of the antigenic suspension (An
et al., 2007; Gaspar et al., 2021). Fig. S3a shows a decrease in the anodic
and cathodic peaks after the deposition of RPR antigen on the MPTS sur-
face (iPA = 35.416 ± 0.218 µA). A slight decrease was observed after BSA
adsorption (iPA = 34.398 ± 0.803 µA).
Impedimetric sensors are practical tools for identifying analytes of
clinical interest, mainly because of the ease of manipulation and sim-
plicity of data analysis (Leva‐Bueno et al., 2020). This strategy also
allows for detection of a specific analyte by using multiple biorecogni-
tion elements, such as proteins, DNA/RNA, lipids, enzymes, antibod-
ies, or aptamers. Electrochemical biosensors are an excellent option
for the detection of syphilis analytes and diagnosis of other sexually
transmitted diseases (Ansari et al., 2009; Brodowski et al., 2021).
Sheng et al. (2010) have developed an impedimetric sensor platform
based on a thiol‐modified probe and enzymatically polymerized polyani-
line. This sensor detects the specific polA gene fragment of syphilis DNA
with a limit of detection of 0.5 pM. Schlichtiger et al. (2013) have assem-
bled 11‐mercaptoundecanoic acid, cardiolipin, and β2‐glycoprotein I on
an SPR gold disk, thus providing an optical sensor with good sensitivity.
Aizawa et al. (2001) have reported a platform comprising an antibody‐
immobilized latex bead suspension in a quartz crystal microbalance,
which can detect agglutination between T. pallidum and the antigenic
latex beads. Runina and coworkers (2018) have created an immunochip
for syphilis detection using multiple recombinant antigens such as Tp15,
Tp17, and Tp47 lipoproteins; the periplasmatic protein Tp0277; and
new synthetic proteins. The proposed platform can detect specific IgG in
serological screening with good sensitivity and specificity.
Charge transfer resistance (RCT) from fitted impedance results was
the main parameter used herein to evaluate the assembly of the sensor
platform and the analyte detection. An increase in RCT is associated
with the interaction between MPTS and RPR antigen (Duncan et al.,
2018). A Randles modified circuit (inset, Fig. S3b) was used to evalu-
ate the electrical kinetics and diffusion processes (Grossi and Riccò,
2017). The circuit is composed of a solution resistance (RS), a constant
phase element (CPE) as a capacitor in parallel with the RCT, and the
Warburg impedance (ZW) (Kokkinos et al., 2016).
The impedance data were fitted on the basis of a Randles modified
circuit (solid black line in the impedance spectra). RCT values are
intrinsically associated with binding events and with changes in the
semicircle diameter. Thus, Rct was chosen as the first element evalu-
ated in the electrochemical biosensor. RS and ZW show insignificant
changes after binding (Nguyen et al., 2020). BGE showed a small semi-
circle (RCT = 0.251 ± 0.07 kΩ) associated with ion diffusion toward
the electrode (Fig. S3b, black curve). MPTS deposition subsequently
significantly increased the RCT (0.988 ± 0.12 kΩ), as shown in
Fig. S3b. The interaction with RPR increased the RCT (Fig. S3b, blue
curve), owing to the decreased electron flow (RCT = 1.70 ± 0.10 kΩ).
3.4. Electrochemical characterization of the recognition capability of the
biosensor
CV and EIS techniques were used to evaluate the binding events
between the RPR‐modified biosensor and anti‐C antibodies (Fig. 3).
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Current Research in Biotechnology 4 (2022) 58–65
The sensor platform was used to analyze healthy human serum as a
negative control to evaluate the sensor’s selectivity. Several positive
samples were prepared in PBS (pH 7) and incubated on the MPTS_RPR
biosensor. A decrease associated with the titer dilution (1:8, 1:16,
1:64, 1:256, 1:512, and 1:1024) was observed after the recognition
process in both anodic and cathodic peaks. The anodic current varia-
tion (ΔI) was estimated according to the following equation (de
Miranda et al., 2017):
ðIb  IaÞ
ΔIð%Þ ¼
Ib
where Ib and Ia correspond to the anodic peak current before and after
target recognition, respectively. ΔI values revealed a gradual decrease
in the highest titer (1:1024) of the target analyte present in the sample
(Table S2).
The Nyquist impedance plots for anti‐C antibody detection in three
syphilitic serum samples (ten measurements) is shown in Fig. 3b. The
data on the equivalent circuit values obtained from the impedimetric
fitting curves are shown in Table 1. The results in Table 1 were eval-
uated according to the relative variation in RCT (ΔRCT), which was
used as a parameter for the determination of RPR/anti‐C antibody
interaction (Yagati et al., 2018):
RCT ðtargetÞ  RCTðbiosensorÞ
ΔRCT ¼ X100
RCTðbiosensorÞ
where RCT (target) is the RCT value after analyte interaction, and
RCT (biosensor) corresponds to the RCT value of the MPTS_RPR modified
electrode. ΔRCT (Fig. 4a) was obtained for multiple ranges of anti‐C
antibodies of the sera in PBS with titers from 1:8 to 1:1024 dilution.
These results revealed a linear relationship, wherein ΔRCT increased
with the target antibody dilution. The linear regression equation was
determined from the calibration plot as y = 144.10x–66.77 with a cor-
relation coefficient (R2) of 0.981. The y values correspond to ΔRCT, and
the × values correspond to the dilution (titer) of the tested samples.
The proposed nanostructured system showed a linear sensitivity range
of 8 to 1024 titer with a standard deviation of 6.19% (S/N = 10). The
nanostructured sensor system demonstrated adequate reproducibility
with a relative standard deviation value of 8.41%. The reproducibility
was determined from the standard deviation’s percentage value for ten
independent sensor systems produced with the same protocol and
experimental conditions.
To investigate the selectivity of the sensor platform, we performed
electrochemical experiments under the same conditions previously
described for anti‐C antibody detection. We evaluated the interaction of
the sensor with glycine, glucose, and citric acid as interfering molecules
at concentrations of 100 mM. The normal glycine, glucose, and citric acid
concentrations in human blood are 390 µM L‐1, 3.9 mM L‐1, and 47 µM L‐1,
respectively (Greim, 2002; Liao et al., 2019; Schmidt et al., 2016). In addi-
tion, serum from a healthy baby and pure anti‐DENV3 were also assessed.
ΔRCT and impedimetric spectra for all analytes in the selectivity tests are
shown in Fig. 5a and 5b. The values observed for interfering species
revealed a small interfacial resistance due to non‐specific adsorption. All
tested negative controls showed an insignificant impedance increase
(Fig. 5). In addition, ΔRCT did not exceed 13% for interfering molecules
and ∼ 32% for anti‐DENV3 antibodies. These values were used as a
threshold for differentiating between infected and negative samples. Our
results suggested that the developed biosensor showed selectivity among
clinically relevant molecules.
Previously, some studies have used the degree of surface coating
(θ) (De Souza et al., 2014; Wang et al., 2014) to assess the bioactivity
of the sensor. The degree of surface coating (θ) is associated with the
difference in RCT between the sensing platform and its response to the
analyte detection, as follows:
RCTðrecogÞ
θ¼1
RCTðtargetÞ
E.M.N. Do Egito et al. Current Research in Biotechnology 4 (2022) 58–65

Fig. 3. Cyclic voltammogram (a) and Nyquist plot (b) of the biosensor after exposure to different titers of anti-C antibodies present in syphilitic human serum.

Table 1
Data on the equivalent circuit elements obtained from the fitted impedance results for anti-C antibody recognition by the sensor system.

Modified electrode Syphilitic serum (titer) RCT (kΩ) CPE (µF) RS (kΩ) W

Bare gold electrode – 0.31 ± 0.11 13.4 ± 0.37 0.106 ± 0.079 1.05 ± 0.04
MPTS – 1.03 ± 0.19 3.51 ± 0.28 0.122 ± 0.043 1.01 ± 0.17

RPR antigen – 1.72 ± 0.15 3.49 ± 0.15 0.129 ± 0.012 0.979 ± 0.12

BSA – 1.82 ± 0.10 3.32 ± 0.24 0.128 ± 0.022 0.941 ± 0.03

1:1024 3.79 ± 0.22 2.85 ± 0.15 0.124 ± 0.023 0.915 ± 0.08

Sensor system 1:512 5.14 ± 0.36 2.21 ± 0.13 0.122 ± 0.014 0.860 ± 0.12

1:256 7.59 ± 0.14 2.02 ± 0.36 0.121 ± 0.019 0.844 ± 0.19

1:64 11.44 ± 0.33 1.79 ± 0.24 0.120 ± 0.005 0.741 ± 0.39

1:16 14.18 ± 0.12 1.72 ± 0.21 0.119 ± 0.011 0.720 ± 0.28

1:8 15.95 ± 0.14 1.64 ± 0.17 0.119 ± 0.022 0.672 ± 0.77

Glucose 2.15 ± 0.12 1.44 ± 0.27 0.323 ± 0.019 0.988 ± 0.15

Glycine 2.02 ± 0.21 1.35 ± 0.19 0.335 ± 0.025 0.958 ± 0.23

Citric acid 2.11 ± 0.07 2.07 ± 0.33 0.340 ± 0.007 0.882 ± 0.79
Interfering molecules
Healthy baby serum 2.18 ± 0.35 1.57 ± 0.47 0.344 ± 0.012 0.912 ± 0.31

Anti-DENV3 2.41 ± 0.13 1.99 ± 0.17 0.341 ± 0.021 0.922 ± 0.44

where RCT (target) is the RCT obtained from different anti‐C antibody
titers after interaction with the biosensor, and RCT (recog) is the RCT
for the MPTS_RPR modified electrode. Fig. 4b shows a plot of θ as a
function of the target antibodies in syphilitic sera. As expected, the θ
values increased in proportion to the decrease in antibody dilution.
Table 2 shows the analytical performance of new biosensor strate-
gies with various sensing elements and targets to diagnose syphilis.
VDRL testing requires an average of 1 h for sample pre‐treatment, reac-
tion, and microscopy to assess the microflocculation. In contrast, the
developed sensor requires approximately 5 min to evaluate the detec-
tion results. Patients with new infections and those weeks after initial
syphilis infection show positivity at titers of 1:8 and 1:32, respectively
(Nayak and Acharjya, 2012) (Fig. S2). Of note, our electrochemical
sensor indicated positivity with a titer of 1:1024.
Cardiolipin, the main component of RPR antigen,
comprises ∼ 100% unsaturated linoleoyl fatty acid with four unsatu-
rated 18‐carbon chains prone to oxidation (Ting et al., 2019). The
cardiolipin‐antibody complexes form through the interaction of the
anti‐C antibodies with phosphate and the β‐hydroxyl group connected
via the central glycerol moiety of cardiolipin. The anionic phospho-
lipid composition contributes to electrostatic interactions with anti-
bodies present in syphilis infection (Schlichtiger et al., 2013). The
immunocomplexes formed on the electrode surface hinder the passage
of the current from the redox pair and therefore block the reduc-
tion–oxidation process. The proposed sensor detects anti‐C antibodies
in a small sample volume (only 2 μL of sample is required). The sensor
system exhibited favorable analytical performance parameters, such as
detection, quantification limits, high selectivity, and specificity.
4. Conclusion
We developed an electrochemical RPR‐based biosensor to detect
anti‐C antibodies in human syphilitic serum. Optical analysis revealed
the formation of immunocomplexes between the RPR and specific anti-

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E.M.N. Do Egito et al. Current Research in Biotechnology 4 (2022) 58–65

Fig. 4. ΔRCT (a) and degree of surface coverage (b) as a function of different titers of anti-C antibodies.

Fig. 5. ΔRCT (a) and Nyquist plot (b) for the selectivity study with interfering molecules.

Table 2
Analytical comparison of innovative strategies for syphilis diagnosis.

Recognition element Sensor platform Transducer Limit of Target Reference

detection

Antigen TpN17 Electrochemical thiol-modified DNA and Square wave voltammetry – Anti-syphilis antibodies Ogden et al.,
methylene blue (SWV) 2019
T. pallidum antigen Gold nanoparticles and biotinylated Plasmonic ELISA 0.98 pg mL−1 Treponemal antibodies Nie et al., 2014
acetylcholinesterase
Treponemal membrane – Surface plasmon resonance – Treponemal antibodies Severs et al.,
Protein A (SPR) 1993
Treponemal lipoprotein r17 – Backscattering – Specific IgG to T. pallidum Kussrow et al.,
interferometry (BSI) 2010
Thiol-modified specific Biotinylated probe, streptavidin, HRP and CV and EIS 0.5 pM PolA gene fragment of T. Sheng et al.,
capture probe polyaniline pallidum 2010
RPR antigen MPTS_RPR modified gold surface CV and EIS 1:1024 titer Anti-cardiolipin This work
antibodies

bodies. The MPTS self‐assembled monolayer efficiently immobilized
RPR antigen as a biorecognition element. The changes in the electro-
chemical response were proportional to the titers of anti‐C antibodies
interacting with the RPR antigen, particularly cardiolipin. The pro-
posed biosensor detected a titer of 1:1024 with excellent sensitivity
and specificity. The biosensor was able to identify anti‐C antibodies
from human syphilitic serum in a 5‐minute test. Our results demon-
strate that the developed biosensor is a promising diagnostic alterna-
tive for identifying syphilis in human samples.
CRediT authorship contribution statement
Elton M.N. Do Egito: Methodology, Investigation, Formal analysis,
Writing – original draft. Alberto G. Silva‐Júnior: Methodology, Inves-

63
E.M.N. Do Egito et al.
tigation, Formal analysis, Writing – original draft. Raiza P.S. Lucena:
Methodology, Investigation, Formal analysis, Writing – original draft.
Maria D.L. Oliveira: Methodology, Investigation, Conceptualization,
Writing – review & editing, Supervision, Funding acquisition. César
A.S. Andrade: Conceptualization, Writing – review & editing, Supervi-
sion, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors are grateful for the support from the Brazilian National
Council for Scientific and Technological Development/CNPq (grant
numbers 314894/2018‐7 and 314756/2018‐3). Alberto G. Silva
Júnior thanks CAPES for a Ph.D. scholarship.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.crbiot.2022.01.001.
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