Transcript
Page 1: SELECTIVITY and CROSS-REACTIVITY

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

Recommended