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Beatriz Arévaloa, V. Serafína, J. F. Beltrán-Sánchezb, J. Aznar-Povedab, A. J. García-Sánchezb, J. García-Harob, S. Campuzanoa, P. Yáñez-Sedeñoa and J. M. Pingarróna.
aDepartment of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, Ciudad Universitaria S/N, 28040, Madrid, Spain. E-mail: [email protected] of Information and Communication Technologies (ICT), Technical University of Cartagena, ETSIT, Campus Muralla del Mar, 30202, Cartagena, Spain.
Fertility-related hormones are the main
components that regulate the reproductive cycleFertility-related hormones are involved in
ovulation and pregnancy
HYPOTHALAMUS
PITUITARY GLAND
PRLLH FSH
E2 P4
Gonadotropic
hormones
Sex steroid hormones
GnRH
OVARIES
UTERUS
PROLACTIN (PRL) stimulates milk production in
mammary glands and progesterone synthesis in the
corpus luteum
PROGESTERONE (P4) prepares the uterus for pregnancy
LUTEINIZING HORMONE (LH)
stimulates the formation of the
corpus luteum and ovulation
ESTRADIOL (E2) keeps the oocyte in the
ovaries and acts as a growth hormone for
reproductive organs
+ +ProtG-MBs cAb P4/ P4-HRP
+ +PRL NA-MBs
dAb-HRP
b-cAb
+ +LH NA-MBs
dAb-HRP
b-cAb
+ +ProtG-MBs cAb E2/ E2-HRP
e-
BQ HRPox H2O2
HQ HRPred H2O
i, n
A
t, s
-14-12-10 -2 0 2 4 60
50
100
150
200
250
300
-i, nA
Log([P4], ng mL-1)
0 20 40 60 80 1000
200
400
600
800
1000
-i, nA
[LH], mIU mL-1
-14-12-10 -2 0 2 4
0
200
400
600
800
1000
-i, nA
Log([E2], ng mL-1)
0 20 40 60 80 1000
200
400
600
800
1000
1200
-i, nA
[PRL], ng mL-1
ANALYTICAL CHARACTERISTIC P4, pg mL-1 E2, pg mL-1 LH, mIU mL-1 PRL, ng mL-1
Linear range 38.1 250.0 23.3 50,000.0 1.0 100.0 1.0 100.0
R20.999 0.998 0.999 0.999
EC50 6 1 1.8 0.2 - -
LOD 2.85/1.7* 5.88/6.88* 0.30/0.10* 0.32/0.32**
p (Hill Slope) 0.48 0.04 0.88 0.09 - -
RSD, (n = 10,[hormone]), % 2.20 (10 ng mL-1) 2.26 (5 ng mL-1) 1.80 (50 ng mL-1) 2.66 (25 ng mL-1)
DIRECT COMPETITIVE FORMAT SANDWICH FORMAT
ANALYTICAL CHARACTERISTICS
SELECTIVITY and
CROSS-REACTIVITY
0
200
400
600
800
1000
1200
1400
SPCE SP4CE SPCESP4CE SPCE SP4CE SPCESP4CE
P4 LH E2 PRL
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
-i, nA
0
2
4
6
8
10
0
1
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
8
9
10
11
0
2
4
6
8
10
B/S (P4,
E2); S
/B
(LH
, PR
L)
Man
Wom
an
Bre
astfee
ding
wom
an
Men
opau
sal
wom
an
0
20
40
60
-i, nA
ESTRADIOL
Man
Wom
an
Bre
astfee
ding
wom
an
Men
opau
sal
wom
an
0
20
40
60
80
100
-i, nA
PROLACTIN
Man
Wom
an
Bre
astfee
ding
wom
an
Men
opau
sal
wom
an
0
5
10
15
20
-i, nA
LUTEINIZING HORMONE
Man
Wom
an
Bre
astfee
ding
wom
an
Men
opau
sal
wom
an
0
20
40
60
80
100
120
140
-i, nA
PROGESTERONE
CHI potentiostat
SALIVA SAMPLES
SP4CEMagnet
* at SPdCE/ ** at SPCE
First multiplexed immunoplatform for the amperometric multidetermination of
four fertility-related hormonoes: P4, E2, LH and PRL
Methodology based on the modification of the MBs’ surface with
competitive (P4, E2) or sandwich (LH, PRL) immunocomplexes.
Attractive analytical characteristics and potential to determine
simultaneously the target hormones in saliva samples.
The developed immunoplatform, coupled to a custom designed quadruple
potentiostat fabricated using commercial off-the-shelf components (COTS-based
potentiostat), can be easily exploited to analyze a larger number of samples or
hormones as a POC device for the monitoring of fertility processes
CONCLUSIONS
ACKNOWLEDGEMENTS
ARISE2 Project “Future IoT Networks and Nano-networks (FINe)” ((AEI)/ (FEDER), (UE)) under ID PID2020-116329GB-C22; ATENTO
project (Fundación Séneca, Región de Murcia), under grant 20889/PI/18; LIFE project (Fondo SUPERA COVID-19 funded by Consejo
Superior de Investigaciones Científicas CSIC, Universidades Españolas and Banco Santander); the financial support of RTI2018-
096135-B-I00 (Spanish Ministerio de Ciencia, Innovación y Universidades) and PID2019-103899RB-I00 (Spanish Ministerio de Ciencia
e Innovación); Research Projects, and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-
4349) are gratefully acknowledged.
B. Arévalo and J. Aznar-Poveda acknowledge Predoctoral contracts from the Spanish Ministerio de Economia y Competitividad
(PRE2019-087596 and BES-2017-081061, respectively).
Block diagram showing the
different electronic components
employed (a), three-dimensional
rendering of the designed and
own-manufactured acquisition
board (b), and an overall view of
the constructed device for the
simultaneous determination of P4,
LH, E2 and PRL hormones in
saliva (c).
0
200
400
600
800
1000
1200
1400
SPCE SP4CE SPCE SP4CE SPCE SP4CE SPCE SP4CE
P4 LH E2 PRL
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
-i, n
A
0
2
4
6
8
10
0
1
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
8
9
10
11
0
2
4
6
8
10
Re
lati
on
B/S
(P
4, E
2);
S/B
(LH
, P
RL)
SPCE
SP4CE
WE1: P4WE2: LH
WE3: E2 WE4: LH0
200
400
600
800
1000
1200
1400
SPCE SP4CE SPCE SP4CE SPCE SP4CE SPCE SP4CE
P4 LH E2 PRL
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
0
2
4
6
8
10
0
200
400
600
800
1000
1200
1400
0
200
400
600
800
1000
1200
1400
-i, n
A
0
2
4
6
8
10
0
1
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
8
9
10
11
0
2
4
6
8
10
Rela
tio
nB
/S (
P4
, E2
); S
/B (
LH,
PR
L)
SPCE
SP4CE
WE1: P4WE2: LH
WE3: E2 WE4: LHWE1: P4
WE2: LH
WE3: E2WE4: PRL
COTS-based
potentiostatELISA
E=-0.2 V vs. Ag pseudo-reference electrode
