Article
A Surface Plasmon Resonance Biosensor Based on
Directly Immobilized Hemoglobin and Myoglobin
Georgi Dyankov
1
, Ekaterina Borisova
2,
* , Evdokia Belina
1
, Hristo Kisov
1
, Ivan Angelov
2,3
,
Alexander Gisbrecht
2
, Velichka Strijkova
1
and Nikola Malinowski
1
1
Institute of Optical Materials and Technology, Bulgarian Academy of Sciences, 109 Acad. G. Bonchev Str.,
1113 Sofia, Bulgaria; gdyankov@iomt.bas.bg (G.D.); evdokiyabelina@yahoo.de (E.B.);
hristokisov@iomt.bas.bg (H.K.); vily@iomt.bas.bg (V.S.); malinowski@iomt.bas.bg (N.M.)
2
Institute of Electronics, Bulgarian Academy of Sciences, 72 Tzarigradsko chaussee Blvd., 1113 Sofia, Bulgaria;
ipangelov@orgchm.bas.bg (I.A.); agiz@abv.bg (A.G.)
3
Institute of Organic Chemistry with a Centre of Phytochemistry—Bulgarian Academy of Sciences,
9 Acad. G. Bonchev Str., 1113 Sofia, Bulgaria
* Correspondence: borisova@ie.bas.bg
Received: 30 July 2020; Accepted: 23 September 2020; Published: 29 September 2020
Abstract:
Immobilization of proteins on a surface plasmon resonance (SPR) transducer is a delicate
procedure since loss of protein bioactivity can occur upon contact with the untreated metal surface.
Solution to the problem is the use of an immobilization matrix having a complex structure. However,
this is at the expense of biosensor selectivity and sensitivity. It has been shown that the matrix-assisted
pulsed laser evaporation (MAPLE) method has been successfully applied for direct immobilization
(without a built-in matrix) of proteins, preserving their bioactivity. So far, MAPLE deposition has
not been performed on a gold surface as required for SPR biosensors. In this paper we study the
impact of direct immobilization of heme proteins (hemoglobin (Hb) and myoglobin (Mb)) on their
bioactivity. For the purpose, Hb and Mb were directly immobilized by MAPLE technique on a SPR
transducer. The bioactivity of the ligands immobilized in the above-mentioned way was assessed by
SPR registration of the molecular reactions of various Hb/Mb functional groups. By SPR we studied
the reaction between the beta chain of the Hb molecule and glucose, which shows the structural
integrity of the immobilized Hb. A supplementary study of films deposited by FTIR and AFM
was provided. The experimental facts showed that direct immobilization of an intact molecule
was achieved.
Keywords: SPR biosensor; direct immobilization; heme proteins; MAPLE deposition
1. Introduction
Heme proteins, such as hemoglobin (Hb) and myoglobin (Mb), have been widely used for sensing
non-organic compounds like NO
2
[
1
], NO
2
and H
2
O
2
[
2
,
3
], C
8
H
8
O
3
[
4
], NO [
5
,
6
], CO [
7
,
8
] and HS [
9
].
Reference [
10
] has reported a biosensor with Hb as the recognition element (ligand) for sensing organic
compounds (glucose).
To date, Hb and Mb have been immobilized mostly onto electrochemical transducers.
Electrochemical biosensors possess high selectivity and biological compatibility but the main drawback
is the low voltammetric response because heme groups are deeply buried. One approach to solving
the problem is using mediators—small molecular compounds that are electroactive. However, this
complicates the electrode process of proteins. Another approach is using a promoter—a small molecular
compound which is electroinactive. The effect is orienting proteins toward the electrodes decreasing
the distance between the redox center and the electrodes, therefore enhancing the electron transfer.
Sensors 2020, 20, 5572; doi:10.3390/s20195572 www.mdpi.com/journal/sensors
