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Development of an Immunosensor for PfHRP 2 as a Biomarker for Malaria

Detection

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DOI: 10.3390/bios7030028

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 biosensors
Article
Development of an Immunosensor for Pf HRP 2 as
a Biomarker for Malaria Detection
Aver Hemben 1 , Jon Ashley 1,2 ID
and Ibtisam E. Tothill 1, * ID

1 Surface Engineering and Nanotechnology Institute, Cranfield University, Cranfield,

Bedfordshire MK43 0AL, UK; a.hemben@cranfield.ac.uk
2 Department of Micro- and Nanotechnology, Technical University of Denmark, Produktionstorvet,
2800 Kgs. Lyngby, Denmark; jash@nanotech.dtu.dk
* Correspondence: i.tothill@cranfield.ac.uk; Tel.: +44-0-750-076-6487

Received: 13 June 2017; Accepted: 12 July 2017; Published: 18 July 2017

Abstract: Plasmodium falciparum histidine-rich protein 2 (Pf HRP 2) was selected in this work as
the biomarker for the detection and diagnosis of malaria. An enzyme-linked immunosorbent assay
(ELISA) was first developed to evaluate the immunoreagent’s suitability for the sensor’s development.
A gold-based sensor with an integrated counter and an Ag/AgCl reference electrode was first
selected and characterised and then used to develop the immunosensor for Pf HRP 2, which enables
a low cost, easy to use, and sensitive biosensor for malaria diagnosis. The sensor was applied to
immobilise the anti-Pf HRP 2 monoclonal antibody as the capture receptor. A sandwich ELISA
assay format was constructed using horseradish peroxidase (HRP) as the enzyme label, and the
electrochemical signal was generated using a 3, 30 , 5, 50 tetramethyl-benzidine dihydrochloride
(TMB)/H2 O2 system. The performance of the assay and the sensor were optimised and characterised,
achieving a PfHRP 2 limit of detection (LOD) of 2.14 ng·mL−1 in buffer samples and 2.95 ng·mL−1 in
100% spiked serum samples. The assay signal was then amplified using gold nanoparticles conjugated
detection antibody-enzyme and a detection limit of 36 pg·mL−1 was achieved in buffer samples and
40 pg·mL−1 in serum samples. This sensor format is ideal for malaria detection and on-site analysis
as a point-of-care device (POC) in resource-limited settings where the implementation of malaria
diagnostics is essential in control and elimination efforts.

Keywords: Malaria; Pf HRP 2; parasites; immunosensor; biosensor; nanoparticles

1. Introduction
Malaria is a serious disease that is caused by an Apicomplexan Plasmodium parasite that is
transmitted by adult female Anopheles mosquitoes, which thrive in tropical and subtropical weather [1].
Malaria affects approximately 50% of the world’s population, and causes millions of deaths [2].
According to the latest World Health Organisation (WHO), estimates, released in December 2016,
there were 212 million cases of malaria in 2015, and 429,000 deaths [2]. From this, the African region
accounted for the most global cases of malaria (88%), followed by the South-East Asia Region (10%)
and the Eastern Mediterranean Region (2%). Despite control efforts, the disease continues to affect
productivity, and therefore an effective diagnosis is required for the successful treatment and reduction
of both complications and mortality [2].
The methods available for the detection of malaria include blood film microscopy, immune-
chromatographic tests, and serological tests. Blood film microscopy shows the highest specificity, as it
depends on the detection of Plasmodium parasites in blood circulation, and in some cases is essential for
epidemiological purposes [3]. This assay is known as the gold standard method for malaria diagnosis
despite problems with its field accuracy, unacceptably high false-positive rates, errors in species
identification, and its operator-dependence [4,5]. Alternative methods, such as laser desorption mass

Biosensors 2017, 7, 28; doi:10.3390/bios7030028 www.mdpi.com/journal/biosensors

Biosensors 2017, 7, 28 2 of 14

spectroscopy (LDMS), loop mediated isothermal amplification (LAMP), and flow cytometry (FCM) are
expensive, time consuming, require specialised training, and are characterised by various levels of
sensitivity or specificity in relation to sample quality [6–8]. Levels of parasitemia are not necessarily
correlative with the progression of the disease, particularly when the parasite is able to adhere to blood
vessel walls. Therefore, more sensitive, easy to use diagnostic tools need to be developed in order to
detect low levels of parasitemia in the field [9].
Serological malaria tests are blood tests that picks up the specific malaria antibodies produced by
the immune system [10]. These methods have a specific use, as they are limited to the measurement of
past exposure to the disease. Methods based on parasite nucleic acid detection [11] have shown great
sensitivity and specificity, but require significant infrastructure and training, and are more expensive
than the blood smear method [12]. Methods based on the use of antibodies to recognise parasite
components or biomarkers have also emerged in recent years [13].
Plasmodium falciparum histidine-rich protein 2 (Pf HRP 2) is a 35 kDa protein comprising unique
tandem repeats (Ala-His-His-Ala-Ala-Asp), and is present in the serum of a malaria-infected patient as
a parasite antigen [14,15]. Pf HRP 2 is also present in food vacuole [16], digestive vacuole [17], and
the membrane surface of the infected red blood cells [18]. Pf HRP 2 is produced in large amounts
by the most lethal of malaria parasites, and is specific to Plasmodium falciparum. Other malaria
biomarkers, such as parasite lactate dehydrogenase (pLDH), and/or parasite aldolase, are common
to all Plasmodium species [19]. In addition, Pf HRP2 has been proven to be useful in detecting the
presence of parasites in cases of placental malaria [20]. The significance of Pf HRP 2 has led to a
lateral flow dipstick test [21,22], enzyme-linked immunosorbent assay (ELISA) tests [4], and Western
blotting [23] for the clinical diagnosis of malaria in support of microscopy. The drawbacks of these
techniques are that they are either of low sensitivity, which could lead to inappropriately withholding
treatment from patients with malaria [19], or are as time consuming as the lab-based methods. Recent
literature has reported the development of different sensors for malaria based on biomarker and
antibody detection [24,25]. Therefore, in this work, we investigated the development of a rapid and
highly sensitive sensor based on a screen-printed device, which would enable its use in low-resource
countries. The method selected was based on chronoamperometry, which is well known for its
high sensitivity [26] and ability to amplify a signal using nanotechnology. The use of biomarker
detection related to parasite infection was also implemented in this work through the selection of
Pf HRP 2. The biosensor was then optimised to achieve a sensitive outcome and a capacity to work in
resource-limited settings, and can be combined with other biomarkers for malaria infection detection.

2. Materials and Methods

2.1. Materials
Plasmodium falciparum histidine-rich protein 2 recombinant protein (PIP001), sandwich pair
HuCAL capture monoclonal antibody (HCA 160, IgG1, clone 14971), and detection (HCA 159, IgG1,
clone 14964) monoclonal antibody conjugated to horseradish peroxidase (HRP) were purchased from
AbDSerotec (UK). Phosphate buffered saline tablets (PBS, pH 7.4), PBST (0.05 v/v Tween-20), Tween-20,
microtitre plates, and MaxiSorp (Nunc Immuno), were purchased from Thermo Fischer Scientific
(Hertfordshire, UK). Bovine serum albumin (BSA), phosphate citrate buffer tablets, sodium hydroxide,
potassium chloride (KCl), sodium carbonate, sodium bicarbonate, 3, 30 , 5, 50 -tetramethyl benzidine
hydrochloride hydrate (TMB) (powder), colloidal gold, hydrogen peroxide, 95% ethanol, potassium
ferricyanide [K3 Fe(CN)6 ], and human serum were purchased from Sigma-Aldrich (Dorset, UK). Milk
concentrate blocking solution was purchased from KPL (Gaithersburg, MD, USA). Double-distilled
ultrapure water produced by a Millipore Direct-Q® 3 UV (Millipore; Molsheim, France) was used
for the analysis. All of the chemicals and solvents were of analytical or HPLC grade, and were used
without further purification.
Biosensors 2017, 7, 28 3 of 14

2.2. Sensors Fabrication and Electrochemical Measurements

Screen-printed gold electrodes (SPGE), consisting of a gold working electrode, a carbon counter
and a silver–silver chloride pseudo-reference electrode were fabricated using a procedure similar to
that described by Noh and Tothill [27], and printed using the facilities at DuPont with inks provided
by the company (DuPont Microcircuit Materials, Bristol, UK). Three electrode batches, JD1, JD2a, and
JD2b, were tested. The printing pastes used for JD1 were BQ221 carbon, BQ331 gold, 5880 Ag/AgCl,
and 5036 blue encapsulant (DuPont Ltd. Bristol, UK), produced in 2010. JD2a and JD2b were different
from JD1 in that the carbon ink used was BQ226, but all other inks used were the same as JD1. The JD2a
sensors were from a batch produced in 2013, while the JD2b sensors were freshly produced (2015).
The gold working electrode had a 5 mm diameter, giving a 19.6 mm2 planar area, and was printed on
a graphite ink layer (dried at 120 ◦ C, 30 min).
The electrochemical procedures were conducted using a computer-controlled four channel
Autolab electrochemical analyser multipotentiostat (Metrohm, The Netherlands) throughout, which
allows the simultaneous detection of four sensors. Data capture was through the supplied GPES
version 4.9) software installed onto a personal computer (PC). The sensor edge connectors were from
PalmSens (Provided by Alvatek, Gloucestershire, UK). The electrodes were characterised using cyclic
voltammetry (CV) and chronoamprometry. The CV scans were conducted by using a 100 µL drop
of potassium ferricyanide (K4 Fe(CN)6 3H2 O) at 0.1, 0.5, and 1 mM in 0.1 M KCl, placed onto the
electrode’s surface. Three scans were taken at varying scan rates (10, 20, 50, 70, and 100 mV·s−1 )
relative to the on board Ag/AgCl reference electrodes. The active area of the working electrode was
calculated [28] using the Randles–Sevcik equation [29].
For sample analysis, each of the measurements was carried out in triplicate using a new strip
in a non-deaerated and unstirred solution. For the selection of the optimal constant potential for the
enzymatic reaction (TMB-H2 O2 -HRP), choroamperometry was conducted using a bare screen-printed
gold electrode with buffer solution (50 mM phosphate citrate buffer, pH 5.0, in 0.1 M KCl) and
a substrate (4 mM TMB, 0.06% H2 O2 ) with an antibody-HRP conjugate. Step amperometry was
conducted at a range of potentials from +600 mV to −400 mV within 600 s for the TMB-H2 O2 -HRP
system, in order to achieve the best signal-to-noise ratio. Data plotted from the steady state current
was used to obtain the concentration of the analyte. Following the measurements, the data were copied
to Microsoft Excel for representation.

2.3. SEM and AFM Scan of the SPGE

Scanning electron microscope (SEM) (Phillips, Guildford, UK) was used to visualise the surface
structure of the gold working electrode at 50× and 3500× magnification. An electron emission
spectrum was also obtained by using the Environmental Scanning Electron Microscope (ESEM) to
determine the composition of the SPGE. Atomic force microscopy (AFM) (Digital instruments, Boston,
MA, USA) was used to obtain the sensor’s surface topography at 25 and 50 mu magnification.

2.4. Immunoassay Development (ELISA)

ELISA tests were first developed using micro well polystyrene plates, MaxiSorp (Nunc
Immuno). A direct assay was first developed by adapting the standard ELISA AbDSerotec protocol
(Abdserotec.com). Following optimisation, Pf HRP 2 recombinant protein (PIP001) was serially diluted
in sodium bicarbonate buffer (pH 9.6) to yield concentrations of 0.01, 0.1, 0.5, 1.0, 5.0, 10, and
100 µg·mL−1 , then 100 µL of the antigen solution was added in triplicate to the plate and incubated
at 4 ◦ C overnight. The control wells contained no antigen. The plate was aspirated and washed
three times using 200 µL 0.01 M PBS Tween-20 (0.05 v/v). A 200 µL of 1% BSA was then used to
block the plate by incubating at 37 ◦ C for 2 h in a Labsystems iEMS Incubator/shaker (Bradenton, FL,
USA). The wash steps were repeated. A detection antibody conjugated with horseradish peroxidase
(HCA 159, 10 µg·mL−1 ) was added to the wells, and incubated at 37 ◦ C for 2 h. The plate was washed
Biosensors 2017, 7, 28 4 of 14

three times in PBS Tween-20 (0.05 v/v). A 100 µL solution of 3, 30 , 5, 50 Tetramethylbenzidine/H2 O2

was added to the reaction wells, and incubated at room temperature for 15 min in the dark. The
reaction was stopped using 50 µL of 1 M H2 SO4 , and read at 450 nm on a Varioskan plate reader
(Thermo Fischer Scientific (Hertfordshire, UK).
A Sandwich ELISA assay was then developed, where 100 µL of Pf HRP 2 capture antibody
(HCA 160) was dissolved in 900 µL of 0.1 M sodium bicarbonate buffer (pH 9.6) to give a concentration
of 50 µg·mL−1 . Twenty microliters (20 µL) of this solution were deposited in a microtiter plate and
incubated overnight at 4 ◦ C. The plate was then washed three times using 200 µL of 0.1 M PBS
Tween-20 (0.05 v/v). Two hundred microliters (200 µL) of 1% BSA was then added to block the plates
by incubating at 37 ◦ C for 2 h. The plate was then washed and 100 µL of serial dilution of the Pf HRP
2 antigen was added to the plate as a 0.01, 0.5, 0.1, 1.0, 10, 50, and 100 µg·mL−1 antigen diluted in
PBS (0.01 M). The control wells contained no antigen. The plate was then incubated at 37 ◦ C for 2 h.
The plate was washed three times in PBS Tween-20 (0.05 v/v). Two hundred microliters (200 µL) of
Pf HRP 2 detection antibody-HRP (HCA 159) were dissolved in 800 µL of PBST-20 (0.05 v/v) to give
a concentration of 20 µg·mL−1 , and incubated at 37 ◦ C for 2 h. The plate was washed three times
using 200 µL of 0.01 M PBS Tween-20, (0.05 v/v). One hundred microliters (100 µL) of 3, 30 , 5, 50
Tetramethylbenzidine were added to the reaction wells, and incubated at room temperature for 15 min
in the dark. The reaction was stopped using 50 µL of 1 M H2 SO4 and read at a 450 nm wavelength.
The standard curve and linear regression with the limit of detection were obtained in Microsoft Excel.
The limit of detection (LOD) was calculated as 3 times the SD of the blank measurement plus the
average blank measurement.

2.5. Optimisation of Capture and Detection Antibody on the Sensor’s Surface

A 20 µL capture antibody in concentrations of 10, 20, and 30 µg·mL−1 in sodium bicarbonate buffer
(0.1 M, pH 9.6) was immobilised by physical adsorption on the gold working electrodes (overnight at
4 ◦ C) in humid conditions. Prior to the immobilisation of the antibodies, the sensors were cured at
120 ◦ C and washed using distilled water. After the immobilisation, the sensors were washed twice
using PBST and dried in a gentle flow of nitrogen. The electrode surface was blocked using 100 µL
of 1:10 milk in PBS (0.01 M) to reduce non-specific binding and incubated for 1 h at 37 ◦ C. After each
incubation step, the surface was washed gently with 100 µL PBST. After washing, 30 µg·mL−1 of
Pf HRP 2 antigen prepared in 20 µL of PBS was dropped onto the working electrode and incubated
for 1 h at 37 ◦ C. The surface was washed with PBST again. The detection antibody HCA 159P was
diluted to a working strength of 30 µg·mL−1 in 1:40 milk concentrate and incubated on the sensor
for 1 h. The sensors were then washed and assayed by using a volume of 100 µL of the TMB-H2 O2
substrate, dropped on the sensor surface covering all three electrodes. The current was then measured
using the potentiostat.
The detection antibody concentration was optimised by repeating the above procedure using
the best concentration of capture antibody obtained in the above experiment (20 µL Pf HRP 2 capture
antibody, 20 µg·mL−1 ) in sodium bicarbonate buffer (0.1 M, pH 9.6). The capture antibody was
immobilised, and the sensor washed and blocked as above. A 30 µg·mL−1 Pf HRP 2 antigen was
prepared in 1:10 milk PBS, and 10 µL was dropped onto the working electrode. The detection antibody
was used at different concentrations of 10, 20, and 30 µg·mL−1 in 1:40 milk PBS. The experiment was
repeated as above.

2.6. Standard Curve and Limit of Detection

The Pf HRP 2 antigen was assayed using 20 µL of capture antibody with a concentration of
20 µg·mL−1 in sodium bicarbonate buffer (0.1 M, pH 9.6) immobilised on the gold working electrode
and incubated overnight at 4 ºC. Concentrations of 0, 2, 16, 20, 40, 64, 80, and 100 ng·mL−1 of Pf HRP
2 antigen were prepared in 1:10 milk PBS (0.01 M), and 20 µL of the antigen solution was dropped onto
the working electrode. The detection antibody-HRP was diluted to a working strength of 20 µg·mL−1
Biosensors 2017, 7, 28 5 of 14

in 1:40 milk PBS. The lowest detection limit of the antigen was determined by 3 times the standard
deviation of the blank value plus the average of the blank measurement, and the data were presented
using Microsoft Excel.
Biosensors 2017, 7, 28 5 of 14
2.7. Signal Amplification Using Gold Nanoparticle
2.7. Signal Amplification Using Gold Nanoparticle
Colloidal gold (40 nm) was employed for its large surface area in an attempt to amplify the
Colloidal gold (40 nm) was employed for its large surface area in an attempt to amplify the
sensor’s signal and lower the detection limit of the target protein. The commercial colloidal gold
sensor’s signal and lower the detection limit of the target protein. The commercial colloidal gold
nanoparticles were investigated using different concentrations of blocking buffer for the optimisation
nanoparticles were investigated using different concentrations of blocking buffer for the optimisation
of the Au nanoparticles’ (AuNP) conjugation to the reporter protein. A 1000 µL quantity of gold colloid
of the Au nanoparticles’ (AuNP) conjugation to the reporter protein. A 1000 µL quantity of gold
was taken in a 1.5 mL tube, and 0.1 µL of 0.2 M NaOH was added, adjusting the pH to 9.0 [30]. A 100 µL
colloid was taken in a 1.5−mL tube, and 0.1 µl of 0.2 M NaOH was added, adjusting the pH to 9.0 [30].
1 detection antibody (HCA 159P) was added, and the mixture was shaken
volume
A 100of µLneat 0.1 mg
volume of·mL
neat 0.1 mg·mL−1 detection antibody (HCA 159P) was added, and the mixture
at was
roomshaken
temperature
at room temperatureblocking
for 1 h. The buffer
for 1 h. The dilutions
blocking ofdilutions
buffer 1:5, 1:10,of1:20,
1:5,and
1:10,1:50
1:20,BSAandand
1:501:5
BSA and
1:10 milk in PBS were examined as the blocker after the antibodies’ attachment
and 1:5 and 1:10 milk in PBS were examined as the blocker after the antibodies’ attachment to the to the nanoparticles.
The tube was then
nanoparticles. Theshaken
tube wasat room temperature
then shaken at room fortemperature
1 h in the dark
for 1and
h inspun at 10,000
the dark and spun rpmat for 10 min
10,000
◦
(4 rpm
C). The supernatant
for 10 min (4 °C). was discarded and
The supernatant wasthe pellet re-suspended
discarded and the pelletinre-suspended
70 µL PBS (0.01in 70M) µLtoPBS
obtain
(0.01the
stock
M) toAuNP—conjugated to the detection to
obtain the stock AuNP—conjugated antibody—HRP, which was stored
the detection antibody—HRP, which 4 ◦ C.stored
at was The stock was
at 4 °C.
diluted 1:5 and
The stock was1:10 to produce
diluted 1:5 andthe1:10 amplified
to produce signal. Another batch
the amplified signal.ofAnother
nanoparticles
batch ofwas also prepared
nanoparticles
in was
a way similar
also to theinabove
prepared a way procedure,
similar tobut thebyabove
adding extra horseradish
procedure, but by addingperoxidase
extra enzyme (HRP)
horseradish
peroxidase
after enzyme (HRP)
the antibody-HRP after the antibody-HRP
attachment. Three microliters attachment.
(3 µL) ofThree
HRPmicroliters
(20 mg·mL (3−µL)
1 ) were
of HRP
added(20 to
mg·mL −1) were added to the AuNP antibody-HRP solution and incubated for 1 h at room temperature
the AuNP antibody-HRP solution and incubated for 1 h at room temperature in a shaker. The tube
wasin then
a shaker.
spunTheat tube
10,000wasrpmthenforspun
10 min (4 ◦ C)
at 10,000 rpm
and forthe
10 supernatant
min (4 °C) and the supernatant
discarded. discarded.
The sediment was
The sediment
re-suspended inwas
1 mL re-suspended
distilled water in 1and
mL the
distilled water continued
procedure and the procedure
as used continued
above. Theasprinciple
used above.of the
The principle
developed of the
sensor candeveloped
be seen in sensor
Scheme can1. be seen in Scheme 1.

Scheme 1. Principle of the developed sensor for malaria detection.

Scheme 1. Principle of the developed sensor for malaria detection.

2.8.2.8.
Human Serum
Human SerumAssay
Assay
InIn
order tototest
order testthe
thesensor
sensorfor
formatrix
matrix effect,
effect, a commerciallyavailable
a commercially availablehuman
humanserum
serum sample
sample was was
used and spiked with different concentrations of the biomarker. Tests were then conducted
used and spiked with different concentrations of the biomarker. Tests were then conducted using using 100%
human
100% serum
humansamples following
serum samples the same
following theprocedure as thatasreported
same procedure in Sections
that reported 2.6 and
in Sections 2.7. 2.7.
2.6 and

3. 3.
Results and
Results andDiscussion
Discussion

3.1.3.1.
Characterisation
Characterisationofofthe
theScreen-Printed
Screen-Printed Electrodes
Electrodes
In order to make a comparison of the 3 electrodes (JD1, JD2a, and JD2b), electrochemical
In order to make a comparison of the 3 electrodes (JD1, JD2a, and JD2b), electrochemical
characterisation was conducted using cyclic voltammetry. This was to investigate the performance
characterisation was conducted using cyclic voltammetry. This was to investigate the performance of
of the new carbon ink used in the JD2 electrodes, and also to study the effect of the production year
the new carbon ink used in the JD2 electrodes, and also to study the effect of the production year on
the performance of the sensors. All of the electrodes were stored at room temperature in dark
conditions. The experiments were carried out in the presence of potassium ferricyanide, in different
concentrations (0.1, 0.5 and 1 mM) and at different scan rates (10, 20, 50, 70 and 100 mV·s−1) relative
Biosensors 2017, 7, 28 6 of 14

on the performance of the sensors. All of the electrodes were stored at room temperature in dark
conditions. The experiments were carried out in the presence of potassium ferricyanide, in different
concentrations (0.1, 0.5 and 1 mM) and at different scan rates (10, 20, 50, 70 and 100 mV·s−16)ofrelative
Biosensors 2017, 7, 28 14
to the on board Ag/AgCl reference electrodes. The cathodic and anodic peak current was used to
calculate
to thetheonactive
boardsurface
Ag/AgClarea of theelectrodes.
reference electrodesTheby cathodic
employingandthe Randle–Sevcik
anodic peak current equation
was used [28,29].
to
The ideal ∆E value for a reversible redox reaction of potassium ferricyanide is 56 to 59 mV, and the
calculate the active surface area of the electrodes by employing the Randle–Sevcik equation [28,29].
The ideal the
ratio between ΔE cathodic
value for aand
reversible
anodic redox reaction
peak is of potassium
1 [31,32]. ferricyanide
In practice, however,isthe
56 difference
to 59 mV, and the
is typically
ratio between the cathodic and anodic peak is 1 [31,32]. In practice, however, the difference is
100 mV and higher [32]. The use of 1 mM potassium ferricyanide resulted in the best reproducibility of
typically 100 mV and higher [32]. The use of 1 mM potassium ferricyanide resulted in the best
the redox reaction (Data not shown). Figure 1 shows the characterisation of the different sensors using
reproducibility of the redox reaction (Data not shown). Figure 1 shows the characterisation of the
CV with 1 mM potassium ferricyanide.
different sensors using CV with 1 mM potassium ferricyanide.

Figure
Figure 1. Cyclic
1. Cyclic voltammogramofofthe
voltammogram thedifferent
different electrodes
electrodes atatdifferent
differentscan rates
scan using
rates a 1 mM
using a 1 mM
potassium ferricyanide solution in 0.1 M KCl, n = 3.
potassium ferricyanide solution in 0.1 M KCl, n = 3.
Biosensors 2017, 7, 28 7 of 14
Biosensors 2017, 7, 28 7 of 14

The results
The resultsshowed
showed thatthat
eveneven though
though JD1 produced
JD1 was was produced
in 2010,init performed
2010, it performed
well whenwell when
compared
compared
to to the JD2 electrodes.
the JD2 electrodes. It was alsoItnoted
was also
thatnoted that the
the active active
surface surface
area of thearea
goldofworking
the goldelectrode
working
electrode
(A (Athe
active %) in %)was
activeJD1 in ~10%
the JD1 wasthan
higher ~10%
in the higher than in (Supplementary
JD2 electrodes the JD2 electrodes (Supplementary
information, Table S1).
information,
This could be Table
due to S1). This could bechanges
physical/chemical due totakingphysical/chemical changes taking
place in the inks/polymer place
as the in the
electrodes
inks/polymer
become older. as the electrodes
Compared become
to the JD2 older. Compared
electrodes, JD1 sufferedtofrom
the JD2
lower electrodes, JD1 suffered
reproducibility. from
The change
lower
in reproducibility.
the base carbon ink showedThe change in on
no effect thethebase carbon
sensor’s ink showedBoth
performance. no JD2a
effectand
onJD2b the showed
sensor’s
performance.data,
comparative Bothand JD2a and experiments
further JD2b showed comparative
continued usingdata, and electrodes.
the JD2b further experiments
In order tocontinued
study the
using thepotential
optimal JD2b electrodes. In order to
for the detection study the optimal
system, potential
current signals for the detection
generated system,
from TMB/H 2 Othe current
2 with the
signals generated
HRP-antibody from were
conjugate TMB/H 2O2 with
analysed using thechronoamperometry.
HRP-antibody conjugate The ratiowere
of theanalysed using
signal current
chronoamperometry.
to the background current Theusing
ratiostep
of amperometry
the signal current(−400 mVto the background
to +600 mV) of 4 mM current
TMBusing step
and 0.06%
amperometry
H 2 O2 with and (−400 mV to
without the+600 mV) of
addition of4the
mM TMB and
detection 0.06% H2O2 with
antibody-HRP in a and without
pH 5.0 citratethe addition
buffer, of
0.1 M
the detection
KCl, antibody-HRP
was calculated. in a pH
The results 5.0 citrate
showed that thebuffer,
best0.1M KCl, was
potential calculated.
in this system isThe −0.2results showed
V using JD2
that the best
electrodes, potential
and in this
therefore this was
system is −0.2for
selected V future
using immunosensor
JD2 electrodes, developments
and therefore this was selected
(Supplementary
for future immunosensor
information, Figure S1). developments (Supplementary information, Figure S1).

3.2.
3.2. SEM,
SEM, ESEM
ESEM and
and AFM
AFM of
of Bare
Bare SPGE
SPGE
The
The working
working electrode
electrode of
of the
the screen-printed
screen-printed goldgold sensor
sensor was
was characterised
characterised using
using scanning
scanning
electron
electron microscopy
microscopy(SEM)
(SEM)andandenvironmental
environmental scanning
scanning electron microscopy
electron microscopy(ESEM), which
(ESEM), show
which the
show
composition of the gold electrode’s surface (Figure 2). The SEM scans showed pinholes
the composition of the gold electrode’s surface (Figure 2). The SEM scans showed pinholes in the in the surface
structure, which were
surface structure, whichformed as a result
were formed as aof the printing
result process
of the printing with awith
process rough granular
a rough surface.
granular The
surface.
ESEM analysis (Figure 2C) gives a small figure insert with the average of three spectra’s
The ESEM analysis (Figure 2C) gives a small figure insert with the average of three spectra’s data and data and
indicates
indicates aa high
high percentage
percentage ofof the
the gold
gold ink
ink (~89.2%)
(~89.2%) used
used to
to produce
produce the
the sensors
sensors with
with carbon
carbon (~9.11)
(~9.11)
and
and oxygen
oxygen (~1.69).
(~1.69).

Figure2.2.SEM
Figure SEMof
ofaaJD2b
JD2bbare
bareelectrode,
electrode,(A)
(A)atat500 ×, (B)
500×, (B) at
at 3500 ×, (C)
3500×, (C) ESEM
ESEM surface
surface analysis
analysis of
of JD2b.
JD2b.
Biosensors 2017, 7, 28 8 of 14
Biosensors 2017, 7, 28 8 of 14

Thesurface
The surfaceroughness
roughness of the
of the goldgold electrode
electrode was visualised
was visualised using using
atomicatomic force microscopy
force microscopy (AFM)
at(AFM)
25 andat
50 25
mu and 50 3).
(Figure muThis
(Figure
shows3).theThis shows the
screen-printed screen-printed
gold gold working
working electrode’s electrode’s
topography, similar
topography,
to similar to the SEM scans.
the SEM scans.

Figure3.3.Three-dimensional
Figure Three-dimensional(3D)
(3D)surface
surfacetopography
topographyof
ofaabare
barescreen-printed
screen-printedgold
goldelectrode
electrode(SPGE)
(SPGE)
using AFM at (A) 25 mu and (B) 50 mu.
using AFM at (A) 25 mu and (B) 50 mu.

3.3.
3.3.Development
Developmentofofthe
theImmunoassay
Immunoassay
First, thePfPfHRP
First,the HRP 2 assay
2 assay waswasdeveloped
developed using the the
using microtiter plateplate
microtiter by a direct and a and
by a direct sandwich assay
a sandwich
format in orderintoorder
assay format investigate the suitability
to investigate of the reagents
the suitability for the detection
of the reagents of Pf HRPof2,PfHRP
for the detection before 2,
moving
before
the assaythe
moving to assay
the sensor’s surface.surface.
to the sensor’s The direct assay assay
The direct was conducted
was conductedby immobilising
by immobilising the antigen by
the antigen
physical adsorption
by physical to the
adsorption toplate, and detecting
the plate, and detectingit using the detection
it using antibody-enzyme
the detection antibody-enzyme conjugate. The
conjugate.
sandwich assayassay
The sandwich used the
usedcapture antibody
the capture immobilised
antibody on the plate
immobilised on thesurface, and theand
plate surface, antigen was added
the antigen was
inadded
solution using different
in solution concentrations.
using different The detection
concentrations. antibody-enzyme
The detection was then added
antibody-enzyme was thento complete
added to
the assay. The
complete resultsThe
the assay. for both assays
results are shown
for both assays in arethe Supplementary
shown InformationInformation
in the Supplementary Figures S2 and S3.
Figure
The LOD was calculated as 0.56 −
·mL for1 −
·mL and1
S2 and Figure S3. The LOD was µgcalculated as the
0.56direct
µg·mL ELISA
−1 anddirect
for the 0.89 µg
ELISA for0.89
theµg·mL
sandwich
−1 for
ELISA. The results
the sandwich reveal
ELISA. The that thereveal
results HuCAL thatsandwich
the HuCAL pairsandwich
recognises and
pair interacts and
recognises withinteracts
the malaria
with
protein, and can
the malaria be used
protein, andin the
can development
be used in of theandevelopment
immunosensor. of Noan further development
immunosensor. of the
No further
assay or optimisation
development was conducted
of the assay on thewas
or optimisation ELISA assay, since
conducted theELISA
on the aim ofassay,
the work
sincewas thetoaim
focus
of on
the
the sensor’s
work was todevelopment.
focus on the sensor’s development.

3.4.
3.4.Development
DevelopmentofofPfHRP
PfHRP22Immunosensor
Immunosensor
Capture
Captureantibody
antibodyoptimisation
optimisationwas wasconducted
conductedusing usingaasandwich
sandwichELISA ELISAformat.
format.Concentrations
Concentrations
of 10, 20, and 30 µg · mL −1 were added to the sensor’s surface (20 µL in sodium bicarbonate buffer,
of 10, 20, and 30 µg·mL were added to the sensor’s surface (20 µL in sodium bicarbonate buffer, 0.1
−1

0.1
M,M,pHpH 9.6)
9.6) toto attach
attach totothethesensor
sensorusing
usingphysical
physicaladsorption
adsorption (overnight
(overnight at at 44 ◦°C).
C). The
The electrodes
electrodeswere were
then blocked using 100 µL, 1:10 milk in PBS (0.01 M) for 1 h at 37 ◦ C, and then washed gently using
then blocked using 100 µL, 1:10 milk in PBS (0.01 M) for 1 h at 37 °C, and then washed gently using
PBST
PBSTbuffer. ThePfPfHRP
buffer.The HRP 22antigen
antigenwaswasthen
thendropped
droppedonto ontothe
thesensor’s
sensor’ssurface
surface(20 ·mL−1−1))
µL,3030µgµg·mL
(20µL,
and ◦ ·mL−1−1))
andincubated
incubatedfor for11hh(37 (37 C),
°C),washed,
washed,andandthen
thenthethedetection
detectionantibody-enzyme
antibody-enzyme(20 µL,3030µgµg·mL
(20µL,
was ◦
wasadded
addedand andincubated
incubated(1(1h,h,37 37 C).
°C).The
Theassay
assaywas wasthen
thenfollowed
followedby byadding
addingthe theTMB-H
TMB-H 2O2O22
substrate, and the signal was recorded using a − 200 mV potential. TMB-H
substrate, and the signal was recorded using a −200 mV potential. TMB-H22O22 was chosen as the O was chosen as the
enzyme
enzymesubstrate
substratefor forthetheenzyme
enzymelabel
labelhorseradish
horseradishperoxidase
peroxidase(HRP)’s
(HRP)’s activity
activity determination
determination [33].[33].
Furthermore, TMB has superior detection properties than other systems
Furthermore, TMB has superior detection properties than other systems [34–36]. Figure 4A shows [34–36]. Figure 4A shows that
the ·mL20 −1 for the−1capture antibody. The response increased
thatbest
theconcentration
best concentration was foundwas to be 20 to
found µgbe µg·mL for the capture antibody. The response
linearly against antibody concentration up to about µg·mL
theto20about −1
increased linearly against antibody concentration up the 20concentration level, and level,
µg·mL−1 concentration after this
and
point the response was lower, which indicates the saturation of the sensor’s surface. A 20 µg·mL -1
after this point the response was lower, which indicates the saturation of the sensor’s surface. A 20
concentration of anti Pfof
µg·mL-1 concentration HRP anti2 PfHRP
antibody was chosen
2 antibody wasaschosen
the optimum concentration
as the optimum for the capture
concentration for the
antibody, since it was the best compromise between the response and the cost
capture antibody, since it was the best compromise between the response and the cost of the antibody. of the antibody.
The
Thedetection
detectionantibody
antibodyconcentration
concentrationwas wasthenthenoptimised
optimisedusingusing2020 ·mL−1−1capture
µgµg·mL captureantibody
antibody
immobilised
immobilisedon onthethesensor’s
sensor’ssurface.
surface. The
The detection
detection antibody-enzyme
antibody-enzyme conjugate
conjugate was was tested
tested atat 10,
10, 20,
20,
and 30 µg · mL − 1 in 1:40 milk PBS and 30 µg·mL −1 antigen. The procedure followed was similar to
and 30 µg·mL in 1:40 milk PBS and 30 µg·mL antigen. The procedure followed was similar to that
−1 −1

that listed above. µg·mL −1

listed above. TheThe results
results are are shown
shown in Figure
in Figure 4B, 4B,
withwith
thethe highest
highest signal
signal recorded
recorded at 20
at 20 µg·mL −1 also

also for detection

for the the detection antibody-HRP
antibody-HRP concentration.
concentration.
Biosensors 2017, 7, 28 9 of 14
Biosensors2017,
Biosensors 2017,7,7,28
28 99of
of14
14

The
Theimmunosensor
The immunosensorwas
immunosensor wasthen
was thendeveloped
then developedfor
developed forthe
for thedetection
the detectionand
detection andquantification
and quantificationof
quantification ofPf
of HRP 2.
PfHRP
PfHRP 2.2.The
The
The
optimal capture (20 µg · mL −1 ) and detection antibody (20 µg·mL−1 ) concentrations were used with
optimalcapture
optimal capture(20 (20µg·mL
µg·mL−1−1))and
anddetection
detectionantibody
antibody(20 (20µg·mL
µg·mL−1−1))concentrations
concentrationswere wereused usedwith
withthe the
the JD2 electrodes
JD2electrodes
JD2 electrodesto to conduct
toconduct a calibration
conductaacalibration
calibrationcurve. curve. Different
curve.Different
DifferentPfHRP Pf HRP
PfHRP22antigen2 antigen concentrations
antigenconcentrations
concentrations(0, (0, 2,
(0,2,2,16, 16,
16,20,
20,
20, ng·mL −1 ) prepared in 1:10 milk/PBS (0.01M) as dilution buffer, and then a set of
64,64,
64, 80,80,
80, and
and and100100
100 ng·mL
ng·mL −1) prepared
−1) prepared inin 1:10
1:10 milk/PBS
milk/PBS (0.01M)
(0.01M) as as dilution
dilution buffer,
buffer, and
and then
then aa set set of
of
experiments
experimentswas
experiments wasconducted
was conductedinin
conducted spiked
in spiked
spiked 100%
100%
100% commercial
commercial
commercial human
human
human serum
serum
serum samples.
samples.
samples. The assay
The
The was
assay
assay runrun
was
was in
run
triplicate
intriplicate
in forfor
triplicate allall
for of the
allof measurements.
ofthe
the measurements.
measurements. TheThe
The blank
blank
blank contained
contained
contained nono antigen
no antigen
antigen for
forthe
for thebuffer
the bufferand
buffer andthe
and theserum
the serum
serum
experiments.
experiments.The
experiments. The results
Theresults
resultsofof both
ofboth assays
bothassays
assaysareare shown
areshown
shownin in Figure
inFigure 5. The
Figure5.5.The results
Theresults agree
resultsagree with
agreewith the
withthe range
rangeused
therange used
used
by
by [37]
[37]for
for the
the detection
detection ofofPfHRP
PfHRP 2.2.The
The effect
effectofoftandem
tandem repeats
repeats ininthe
the structure
by [37] for the detection of PfHRP 2. The effect of tandem repeats in the structure of the protein makestructure of
ofthe
the protein
protein make
make
the
theantigen
the antigeneasy
antigen easyto
easy todetect;
to detect;however,
detect; however,the
however, theconcentration
the concentrationof
concentration ofthe
of theanalyte
the analyteisisisinfluenced
analyte influencedby
influenced bythe
by thematrix.
the matrix.
matrix.

Figure4.44 Chronoamperometric
Figure
Figure Chronoamperometric response
Chronoamperometric response of
response of(A)
(A)different
differentconcentrations
concentrationsof
ofcapture
captureantibody
capture antibodyon
antibody onthe
on the
the
sensor’ssurface
sensor’s
sensor’s surfacein
surface inaaasandwich
in sandwichassay
sandwich assayformat;
assay format,(B)
format, (B)different
(B) differentconcentrations
different concentrationsof
concentrations ofdetection-horseradish
of detection-horseradish
detection-horseradish
peroxidase(HRP)
peroxidase
peroxidase (HRP)antibody
(HRP) antibodyin
antibody inaaasandwich
in sandwichassay
sandwich assayformat,
assay format,nnn===3.
format, 3.3.

Figure5.5.5.Linear
Figure
Figure Linearregression
Linear regression
regression analysis
analysis
analysis of the
of chronoamperometric
of the
the chronoamperometric
chronoamperometric response of Pf HRP
response
response 2 detection
of PfHRP
of PfHRP in buffer
22 detection
detection in
in
(PBS, 0.01M, pH 7.4, spiked with 1–100 ng · mL −1 Pf HRP 2) (A) and 100% serum samples (spiked with
buffer (PBS, 0.01M, pH 7.4, spiked with 1–100 ng·mL −1 PfHRP 2) (A) and 100%
buffer (PBS, 0.01M, pH 7.4, spiked with 1–100 ng·mL PfHRP 2) (A) and 100% serum samples (spiked
−1 serum samples (spiked
1–100
with ng
with ·mL−
1–100
1–100
1 Pf HRP
ng·mL
ng·mL 2) (B) on
PfHRP
−1−1 PfHRP 2)2) a(B)
JD2on
(B) sensor.
on aa JD2
JD2Measurements were conducted
sensor. Measurements
sensor. Measurements were at
were −0.2 V. Correlation
conducted
conducted at −0.2
at −0.2 V.
V.
coefficient and R2 value of 0.9827 for the buffer matrix and 0.98 for the serum matrix (n = 3).
Correlationcoefficient
Correlation coefficientand 2 valueof
andRR2 value of0.9827
0.9827forforthe
thebuffer
buffermatrix
matrixand
and0.98
0.98for
forthe
theserum
serummatrix
matrix(n (n==
3).
3).
From the data shown in Figure 5A, the limit of detection (LOD) for the buffer samples was
Fromas
From
calculated the
the data
data
2.14 ·mL−1 ,in
shown
ngshown in Figure
Figure
and 5A,spiked
5A,
for the the limit
the limit of detection
of
human detection (LOD) for
(LOD)
serum samples for the buffer
the
(Figure buffer
5B) the samples
samples
LOD was was
was
calculated
calculated
2.95 ng·mLas −as.2.14
1 2.14
Theseng·mL
ng·mL −1, and for the spiked human serum samples (Figure 5B) the LOD was 2.95
−1 , and
results for the
show thatspiked humangive
both assays serum samples
a similar (Figure 5B)
detection thebut
limit, LOD was
with 2.95
lower
ng·mL−1−1. .achieved
ng·mL
readings Theseresults
These results show
show
for the serum that
that bothassays
both
samples. assays give
Thisgive aasimilar
is very similar detection
detection
encouraging limit,
andlimit, butwith
but
indicates with lower
that lower
the readings
readings
sensors are
achieved
achieved
able forthe
for
to perform the serum
serum
well samples.
samples.
using Thisisisvery
This
100% commercial veryhuman
encouraging
encouraging
serum and andindicates
indicates
samples. thatthe
The that thesensors
difference sensors
in matrix areable
are ableto
affectsto
perform
perform well
well using
using 100%
100% commercial
commercial human
human serum
serum samples.
samples. The
The difference
difference
the rate of electron exchange, which occurs when the analyte is oxidized when a potential difference in
in matrix
matrix affects
affects the
the rate
rate
isofapplied.
of electronThe
electron exchange,
exchange, which occurs
which
lower readings occurs when
can be when
due the
tothe
small analyte
analyte isis oxidized
proteins oxidized whenattaching
when
in the samples aa potential
potential difference
to difference
the sensor’s isis
applied.
applied.
surface and The
The lower readings
lower
affecting readings can be
can
the electron be due
due to
transfer. to small
small
The proteinsand
proteins
sensitivity in the
in the samples
samples attaching
reproducibility attaching toto the
of the sensorsthe sensor’s
sensor’s
in this
surfaceand
surface andaffecting
affectingthetheelectron
electrontransfer.
transfer.The
Thesensitivity
sensitivityand andreproducibility
reproducibilityof ofthe
thesensors
sensorsin inthis
this
assayare
assay areshown
shownto tobebeadequate
adequatefor forthe
thedetection
detectionof of the
themalaria
malariabiomarker,
biomarker,sincesinceaablood
bloodlevel
levelof of~~
9.45ng·mL
9.45 ng·mL−1−1has hasbeen
beenreported
reportedto tobebePlasmodium
Plasmodiumsp. sp.specific
specificandandmalaria
malariapositive
positive[38–40].
[38–40].
Biosensors 2017, 7, 28 10 of 14

assay are
Biosensors shown
2017, 7, 28 to be adequate for the detection of the malaria biomarker, since a blood level 10 of of
14
~9.45 ng·mL−1 has been reported to be Plasmodium sp. specific and malaria positive [38–40].
investigate if we can
To investigate can improve
improve the
the detection
detection limit further, the the detection
detection antibody-HRP
antibody-HRP was
nanoparticles (40 nm)
attached to gold nanoparticles nm) and
and used
used in in the
the sandwich
sandwich assay.
assay. Several optimisation
optimisation
experiments were wereconducted
conductedtotoachieve
achievethethe
best results
best resultsbefore a calibration
before curve
a calibration was repeated
curve with
was repeated
serialserial
with dilution of the
dilution of antigen in buffer
the antigen first,
in buffer and
first, andthenthenthe
theexperiments
experimentswerewererepeated
repeated in
in 100%
commercial human human serum
serum samples.
samples.TheThefull
fullprocedures
procedures are listed
are in in
listed Section 2.7.2.7.
Section Figure 6 shows
Figure the
6 shows
linear regression of the results achieved using the amplified
the linear regression of the results achieved using the amplified assay with the assay with the gold nanoparticles
nanoparticles
conjugated detection
detectionantibody-HRP
antibody-HRPalone,
alone,without
without thetheaddition
additionof free enzymes
of free enzymes to attach to thetogold
to attach the
nanoparticles.
gold nanoparticles.

Figure 6.
6. Linear
Linearregression
regressionofofthethe
chronoamperometric
chronoamperometric response
responseof PfHRP 2 detection
of Pf HRP in buffer
2 detection (PBS,
in buffer
0.01M,0.01M,
(PBS, pH 7.4,
pH spiked withwith
7.4, spiked 0.05–0.5 ng·mL
0.05–0.5 ng·mL −1 Pf HRP
−1 PfHRP 2) (A) and
2) (A) and100
100%%spiked
spiked serum
serum samples
(Commercial human
(Commercial human serum
serum samples
samples spiked
spiked with
with 0.05–0.5
0.05–0.5 ng ·mL−
ng·mL −11PfHRP 2) (B) on JD2 sensors using
Pf HRP 2) (B) on JD2 sensors
gold nanoparticles
nanoparticles conjugated
conjugatedto tothe
thedetection
detectionantibody-HRP.
antibody-HRP. Measurements
Measurements were were conducted
conducted at −at0.2
−0.2
V.
V. Correlation
Correlation coefficient
coefficient andand2
R R 2 value
value of 0.955
of 0.955 forfor
thethe buffer
buffer matrix
matrix andand 0.9844
0.9844 forfor
thethe serum
serum (n =(n3).
= 3).

From the
From the above
above data,
data, an
an LOD
LOD of of 36
36pgpg·mL
·mL−1 was
−1
was obtained
obtained in in the
the amplified
amplified buffer
buffer samples with
samples with
an R 2 value of 0.955 (Figure 6A). An LOD of 40 pg·mL−1 was also obtained in the amplified 100%
an R2 value of 0.955 (Figure 6A). An LOD of 40 pg·mL−1 was also obtained in the amplified 100%
serum samples
serum samples (Figure
(Figure 6B).
6B). The
The AuNP
AuNP results
results gave
gave excellent
excellent sensitivity and limit
sensitivity and limit of
of detection
detection without
without
the use of additional free enzymes (horseradish peroxidase) to load the particles. The
the use of additional free enzymes (horseradish peroxidase) to load the particles. best AuNP
The best AuNP
conjugate stock dilution used was 1:10 milk in PBS, and also blocking with milk proteins. The serum
conjugate stock dilution used was 1:10 milk in PBS, and also blocking with milk proteins. The serum
proteins in
proteins in the
the samples
samples inin this
this assay
assay also
also showed
showed aa similar
similar trend
trend to to the
the non-amplified
non-amplified assay, in that
assay, in that
similar responses where achieved to the buffer samples. Table 1 shows some of the
similar responses where achieved to the buffer samples. Table 1 shows some of the different biosensor different biosensor
technology for
technology detecting PfHRP
for detecting PfHRP 2, 2, and
and their
their detection
detection limits.
limits. These
These areare comparable
comparable to to the limits
the limits
achieved using our sensor. Our work showed that a proof of concept sensor
achieved using our sensor. Our work showed that a proof of concept sensor was developed and this was developed and this
can achieve a much lower detection limit than is required for malaria’s positive
can achieve a much lower detection limit than is required for malaria’s positive detection in blood
detection in blood
samples. Further work will be done to examine the sensor in patient serum samples to confirm the
samples. Further work will be done to examine the sensor in patient serum samples to confirm the
results achieved in this work.
results achieved in this work.
Table 1. Different biosensor technology for detecting PfHRP 2, and their detection limits.
Table 1. Different biosensor technology for detecting PfHRP 2, and their detection limits.
Analyte Assay principle Range Detection limit References
Analyte Assay Principle Range Detection Limit References
PfPfHRP
HRP 22 SPR
SPR - - 5.6 pg·mL −1
5.6 pg ·mL−1 [25]
[25]
Carbon
CarbonSPE
SPEmodified
modifiedwith
with −1
PfPfHRP
HRP 22 - - −1 ·mL
8 ng
8 ng·mL [37]
[37]
MWCN
MWCN andAu/MWCN
and Au/MWCN
Carbon
CarbonSPE
SPEmodified
modifiedwith
with
PfPfHRP
HRP 22 - - - - [41]
[41]
AuNPs/Al
AuNPs/Al2 2O
O33sol–gel
sol–gel
Graphite–epoxy
Graphite–epoxycomposite
composite −1
PfPfHRP
HRP 22 - - −1 ·mL
0.36 ng
0.36 ng·mL [42]
[42]
magneto
magnetoelectrodes
electrodes
Polydimethylsiloxane
PfHRP 2 - 16 ng·mL−1 [43]
microfluidic chips
Carbon nanofiber forest 0.01–10
PfHRP 2 0.025 ng·mL−1 [44]
grown on glass microballons ng·mL−1
Biosensors 2017, 7, 28 11 of 14

Table 1. Cont.

Analyte Assay Principle Range Detection Limit References

Polydimethylsiloxane
Pf HRP 2 - 16 ng·mL−1 [43]
microfluidic chips
Carbon nanofiber forest grown on
Pf HRP 2 0.01–10 ng·mL−1 0.025 ng·mL−1 [44]
glass microballons
Mercaptopropylphosphonic acid
10 ag·mL−1 –
Pf HRP 2 functionalized copper doped zinc 6.8 ag·mL−1 [45]
10 µg·mL−1
oxide nanofibers
Low electrocatalytic indium tin
1 pg·mL−1 –
Pf HRP 2 oxide (ITO) on glass electrodes; 2.2 pg·mL−1 [46]
100 ng·mL−1
APTES-glutaraldehyde modified

4. Conclusions
An immunosensor has been successfully developed using a sandwich ELISA assay on JD2 gold
screen-printed electrodes. Milk concentrate was used as the blocking protein, as it reduced non-specific
binding on the electrode surface. With both malaria antigen and antibodies being very expensive, care
had to be taken in designing the experiments to achieve optimised results. An ELISA test was first
developed to check the affinity of both antibodies toward the antigen Pf HRP 2. An immunosensor
was then developed and optimised with electrochemical measurements that produced a 2.14 ng·mL−1
detection limit for the buffer samples, which is better than the ELISA assay developed in this work.
Spiked 100% serum samples also achieved a very good LOD of 2.95 ng·mL−1 . An amplified signal is
also achievable using the sensor with AuNPs conjugated to the detection antibody-enzyme. Signal
amplification using gold nanoparticles gave an LOD of 36 pg·mL−1 , while the serum assay gave an
LOD of 40 pg·mL−1 . The developed immunosensor offers a highly sensitive, portable, and low cost
method of detecting Plasmodium falciparum histidine-rich protein 2. Future experiments will look at
real samples analysis using patient serum samples.

Supplementary Materials: Figure S1: Optimum potential determination by step potential of TMB/H2 O2 system
with antibody-HRP on JD2 electrodes. The results shown are after subtracting the signal with no enzyme, Figure S2:
(A) Standard curve of absorbance versus antigen concentration in a direct ELISA assay, (B) linear regression with
correlation coefficient and R2 value of 0.9612, limit of detection is 0.56 µg·mL−1 , Figure S3: (A) Standard curve
of absorbance versus antigen concentration in a Sandwich ELISA assay, (B) linear regression with correlation
coefficient and R2 value of 0.9755. Limit of detection is 0.89 µg·mL−1 , Table S1: Overview of cyclic voltammetric
analyses of the three electrodes, JD1, JD2a and JD2b at 20 mV·s−1 , using 1 mM potassium ferricyanide solution in
0.1 M KCl, n = 5.
Acknowledgments: The authors would like to express their thanks to DuPont Microcircuit Materials, UK, for the
screen-printed electrodes.
Author Contributions: Aver Hemben performed all the experimental work and wrote the draft of the paper;
Jon Ashley assisted in the laboratory work; Ibtisam E. Tothill supervised and directed the research and finalized
the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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