Citation: Munteanu, I.G.; Apetrei, C.
Tyrosinase-Based Biosensor—A New
Tool for Chlorogenic Acid Detection
in Nutraceutical Formulations.
Materials 2022, 15, 3221. https://
doi.org/10.3390/ma15093221
Academic Editor: Daniela Iannazzo
Received: 22 February 2022
Accepted: 28 April 2022
Published: 29 April 2022
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Article
Tyrosinase-Based Biosensor—A New Tool for Chlorogenic Acid
Detection in Nutraceutical Formulations
Irina Georgiana Munteanu and Constantin Apetrei *
Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment, Dunărea de Jos
University of Gala¸ti, 47 Domneasca Street, 800008 Gala¸ti, Romania; georgiana.munteanu@ugal.ro
* Correspondence: apetreic@ugal.ro; Tel.: +40-727-580-914
Abstract:
The purpose of our research was to develop a new enzymatic biosensor, GPH-MnPc-
Tyr/SPE, using as a support screen-printed carbon electrode (SPE) modified with graphene, man-
ganese phthalocyanine, and tyrosinase, with the aim of developing sensitive detection of chlorogenic
acid (CGA). To immobilise tyrosinase on the sensor surface, crosslinking with the glutaraldehyde
technique was used, thus increasing the enzyme bioactivity on this electrode. The modified elec-
trode has a great catalytic effect on the electrochemical redox of chlorogenic acid, compared to the
simple, unmodified SPE. The peak current response of the biosensor for CGA was linear in the
range of 0.1–10.48
µ
M, obtaining a calibration curve using cyclic voltammetry (CV) and square-wave
voltammetry (SWV). Subsequently, the detection limit (LOD) and the quantification limit (LOQ)
were determined, obtaining low values, i.e., LOD = 1.40
×
10
−6
M; LOQ = 4.69
×
10
−6
M by cyclic
voltammetry and LOD = 2.32
×
10
−7
M; LOQ = 7.74
×
10
−7
M, by square-wave voltammetry (SWV).
These results demonstrate that the method is suitable for the detection of CGA in nutraceutical
formulations. Therefore, GPH-MnPc-Tyr/SPE was used for the quantitative determination of CGA
in three products, by means of cyclic voltammetry. The Folin–Ciocalteu spectrophotometric assay
was used for the validation of the results, obtaining a good correlation between the voltammetric
method and the spectrophotometric one, at a confidence level of 95%. Moreover, by means of the
DPPH method, the antioxidant activity of the compound was determined, thus demonstrating the
antioxidant effect of CGA in all nutraceuticals studied.
Keywords: chlorogenic acid; biosensor; manganese phthalocyanine; tyrosinase
1. Introduction
Phenolic compounds are considered to be substances beneficial for human health
through exercising various biological effects, such as scavenging free radicals, modu-
lating enzymatic activity, metal chelation, and the modification of signal transduction
pathways [
1
]. Epidemiological studies have demonstrated the relationship between the
intake of foods containing a large amount of phytochemicals and minimising the risk of
developing some diseases, such as cancer, coronary diseases, and osteoporosis, and the
results of these studies have promoted interest in consuming phenolic compounds from
various sources [2].
Phenolic compounds are mainly classified into phenolic and flavonoid categories. An
important class within the category of phenolic acids is represented by hydroxycinnamic
acids, and chlorogenic acid (CGA) is their main compound [
3
]. Chemically, chlorogenic
acid is the ester of caffeic acid and quinic acid [4].
CGA is widely present in many vegetable products, such as apples [
5
], potatoes [
6
],
coffee beans [
7
], grapes [
8
], plums [
9
], honeysuckle [
10
], wormwood [
11
], cabbage [
12
]
and kiwi [
13
]. It was noticed that CGA has antioxidant [
14
], anti-inflammatory [
14
], anti-
hypertensive [
15
], anti-diabetic [
16
], anti-neurodegenerative [
17
] and antitumoral proper-
ties [
18
]. Moreover, most research on the benefits of CGA for human health was carried
Materials 2022, 15, 3221. https://doi.org/10.3390/ma15093221 https://www.mdpi.com/journal/materials
