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\begin{document}

\title{Fluorescent optrode for proteins based on a diketopyrrolopyrrole
derivative: practical application to total protein determination in
urine}



\author[1]{Marta Veríssimo}%
\author[1]{Vitor Almovodar}%
\author[1]{Augusto Tomé}%
\author[1]{Maria Teresa Seabra dos Reis Gomes}%
\affil[1]{University of Aveiro}%


\vspace{-1em}



  
  \date{May 04, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
A fluorescent sensor based on a diketopyrrolopyrrole (DPP) derivative
for protein detection was designed and tested in urine samples. The DPP
derivative emits visible light as a solid, making this molecule
particularly appealing as a recognition molecule for a sensing optical
fibre. In the presence of proteins, the sensor displayed a fluorescence
enhancement. The sensor linear dynamic range for protein ranges from 10
mg L-1 to 100 mg L-1. Detection limit (LOD) of this fluorescent optrode
was better than the one of the classical spectrophotometric Biuret
method and similar to the LOD of the spectrophotometric Ponceau-S/TCA
method. Economy of reagents and time is a clear advantage of the
analytical method based on the optrode we are now proposing, regarding
the Ponceau-S/TCA, thanks to the reversibility of recognition layer, and
to the needleless of treating the standards with trichloroacetic acid,
and consequently of centrifuging them. Besides, the most common
interferences in protein determination by classical spectrophotometric
methods, like sodium dodecyl sulfate, acetone, and glucose, do not
affect the optrode response, neither does fluoride ion. The new
fluorescent optrode was tested in the determination of total proteins in
urine from healthy individuals (low concentrations). Results were not
statistically different from the ones obtained with the Ponceau-S/TCA
spectrophotometric method.%
\end{abstract}%



\sloppy


\textbf{Abstract}

A fluorescent sensor based on a diketopyrrolopyrrole (DPP) derivative
for protein detection was designed and tested in urine samples. The DPP
derivative emits visible light as a solid, making this molecule
particularly appealing as a recognition molecule for a sensing optical
fibre. In the presence of proteins, the sensor displayed a fluorescence
enhancement. The sensor linear dynamic range for protein ranges from 10
mg L\textsuperscript{-1} to 100 mg L\textsuperscript{-1}. Detection
limit (LOD) of this fluorescent optrode was better than the one of the
classical spectrophotometric Biuret method and similar to the LOD of the
spectrophotometric Ponceau-S/TCA method. Economy of reagents and time is
a clear advantage of the analytical method based on the optrode we are
now proposing, regarding the Ponceau-S/TCA, thanks to the reversibility
of recognition layer, and to the needleless of treating the standards
with trichloroacetic acid, and consequently of centrifuging them.
Besides, the most common interferences in protein determination by
classical spectrophotometric methods, like sodium dodecyl sulfate,
acetone, and glucose, do not affect the optrode response, neither does
fluoride ion.

The new fluorescent optrode was tested in the determination of total
proteins in urine from healthy individuals (low concentrations). Results
were not statistically different from the ones obtained with the
Ponceau-S/TCA spectrophotometric method.

\textbf{Keywords:} protein, fluorescence sensor, diketopyrrolopyrrole,
optical fibre sensor, urine

1. Introduction

All living organisms, large or small, plant or animal, are made of
cells, which are partly made of proteins. Proteins, also known as
polypeptides, are organic compounds made up of amino acids. Twenty known
amino acids link together to form various kinds of protein, which are
arranged in a linear chain and folded, to form three-dimensional
molecules with complex shapes. Proteins may differ in the type, number,
and sequence of amino acids that make up the polypeptide bone. Having
different molecular structures, nutritional attributes, and
physicochemical properties may also be diverse. Among their many
functions, proteins catalyse reactions, transport oxygen and defend
organisms from infection. They are crucial building blocks of organisms,
they package up the DNA in chromosomes, and they insulate the cells of
the nervous system. Proteins participate in virtually every process
within cells. In short, protein matters!

Protein quantification is necessary in many different fields, such as
food science and technology, biochemistry, physiology, molecular and
protein biology, protein chemistry, clinical analysis, and other
research applications. The careful selection of the protein
quantification method according to the type of sample and the purpose of
the analysis is required to ensure accurate data.

A large number of methods have been developed to quantitate complex
mixtures of proteins either directly or indirectly (M\selectlanguage{ngerman}æhre, Dalheim,
Edvinsen, Elvevoll, \& Jensen, 2018). Direct protein determination is
based on the analysis of amino acid residues. A protein sample is first
hydrolysed (\emph{e.g.} using a strong acid) to release the amino acids,
which are then separated by ion exchange, affinity or absorption
chromatography. The protein content is calculated as the sum of
individual amino acid residues after subtraction of the molecular mass
of H\textsubscript{2}O. The major drawback of this method is that the
hydrolyses of peptide bonds could lead to the destruction of some amino
acids, which may result in protein underestimation. High costs could
also prevent the use of this method by food science laboratories,
despite being the AOAC recommended method for food proteins (FAO, 2003).

Protein determination can be indirectly inferred by the nitrogen
content, or after chemical reactions with functional groups within the
protein(Mæhre et al., 2018). Two methods are currently used for
determining total proteins inferred by the nitrogen content: the
Kjeldahl method and the Dumas method. In the Kjeldahl method, the sample
is digested with sulfuric acid so that it releases nitrogen in the form
of ammonium sulfate, which is converted into ammoniac by treatment with
sodium hydroxide, and further quantified by a suitable titration after
being trapped in an acid solution. The amount of protein present is
calculated from the nitrogen concentration found. Because this method
does not measure the protein content directly, a conversion factor (F)
is needed to translate the measured nitrogen concentration into a
protein concentration. A conversion factor of 6.25~(equivalent to 0.16 g
nitrogen per gram of protein) is used for many applications, although
the conversion factor depends on the amino acid composition. Although
AOAC International considers acceptable the Kjeldahl method for
determination of protein based on total N content when data on amino
acids analyses are not available (FAO, 2003), it has been proven that
the average value factor of 6.25 may lead to errors in the protein
content estimation (Mæhre et al., 2018), and several species-specific
conversion factors have been suggested in the literature. Besides, the
use of concentrated sulfuric acid at high temperatures poses a
considerable hazard, requires skilled staff and the technique is tedious
and time-consuming to carry out.

In the Dumas method, the sample is combusted in a high temperature
(about 900 ºC) chamber in the presence of oxygen. The released gases
passed through special columns that absorb CO\textsubscript{2} and
H\textsubscript{2}O, and the nitrogen content is afterwards measured by
passing the remaining gases through a column that has a thermal
conductivity detector at the end. Like in the Kjeldahl method, it is
necessary to convert the concentration of nitrogen to protein, using a
suitable conversion factor. This method is much faster than the Kjeldahl
method, does not need toxic chemicals nor catalyst, and many samples can
be measured automatically. However, it has a high initial cost, and,
just like the previous method, different proteins need different
correction factors.

Besides, none of these methods distinguishes between protein-based
nitrogen from non-protein nitrogen. Nitrogen from non-protein additives
or contaminants in the food, such as the fraudulent addition of melamine
to milk, is also measured, and exploitation of this analytical
vulnerability has led to serious incidents in the past (Moore, Devries,
Lipp, Griffiths, \& Abernethy, 2010).

Indirect protein determination after chemical reactions with functional
groups within the proteins comprise spectrophotometric methods such as
the direct measurement of UV absorbance at 280 nm, Biuret, Lowry
(Folin-Ciocalteau), Smith (Bicinchoninic acid), Bradford (Coomassie
Brilliant Blue) and, Pesce and Strande (Ponceau-S/TCA) assays, just to
point out the six spectrophotometric methods mostly used (Dilena,
Penberthy, \& Fraser, 1983; Mæhre et al., 2018; Okutucu, Dinçer, Habib,
\& Figen, 2007; Zaia, Zaia, \& Lichtig, 1998). These six methods will be
presented briefly, along with their advantages and disadvantages.

Simple but often unreliable, the direct ultraviolet absorption estimates
the amount of protein by measuring the characteristic absorption of
aromatic amino acids, mainly tyrosine and tryptophan, at 280 nm. The
sensitivity of the method is moderate but has the advantage of not
requiring special reagents, being simple, and the fact that the method
is non-destructive, allowing to recover valuable proteins. Unless the
protein sample is pure, and its extinction coefficient is known, UV
determination of protein concentration will invariably be prone to
significant errors (Olson \& Markwell, 2007). Other disadvantages
include the requirement of UV spectrophotometers and quartz cuvettes and
the fact that virtually everything, including commonly used buffers,
does absorb in the UV region (Zaia et al., 1998). Furthermore, it is
incompatible with a wide range of protein extraction methods which
frequently employ detergents and denaturing agents.

The Biuret assay is a chemical test for proteins based on the formation
of a coloured chelated complex between cupric ions and peptide bonds,
forming a complex with tartrate, which absorbs at 540-545 nm. The
intensity of the colour, and hence the absorption at 540-545 nm, is
directly proportional to protein concentration, according to the
Beer-Lambert law. A significant disadvantage of this assay is its poor
sensitivity. Reported linear range is between 0.5-4.0 mg
mL\textsuperscript{-1} (Okutucu et al., 2007; Zaia et al., 1998).
Another drawback of this assay is that some compounds used in the
laboratory, such as Tris buffer and ammonium sulfate, as well as
endogenous compounds in crude extracts, can interfere with colour
development or generate coloured complexes themselves. Proteins with an
abnormally high or low percentage of amino acids with aromatic side
groups will give high or low readings, respectively (Saxena, Baunthiyal,
\& Ravi, 2015).

Lowry's protein assay combines the Biuret reagent with another reagent,
the Folin-Ciocalteau phenol reagent. First, copper ions are reduced
under alkaline conditions and form a complex with peptide bonds of the
protein. Second, the complexed tyrosine and tryptophan react with
Folin-Ciocalteau phenol reagent to give an intense blue-green colour,
which can be detected somewhere between 500-700 nm. This method has been
widely used for protein determination for many decades, due to its
simplicity. However, it is susceptible to a wide range of nonprotein
substances generally present in most extracts. Besides, the Lowry's
phenol reagent is unstable in alkaline solution, and the two-step
addition of the colour-developing reagents are cumbersome and tedious
(Choi, Chung, Chang, Chun, \& Lee, 1993; Mæhre et al., 2018). The
absorbance of the reaction mixture is not strictly proportional to
protein concentration, and colour development is slow and fades
relatively rapidly, therefore, reasonable precise reaction times, and
temperatures are required (Zaia et al., 1998). However, Lowry assay is
somewhat more sensitive than the Biuret assay, with a reported linear
range between 0.02-0.5 mg mL\textsuperscript{-1} (Okutucu et al., 2007).

The Smith assay, also known as the Bicinchoninic acid (BCA) protein
assay, has been advertised as an alternative to the Lowry assay. Like
the Lowry assay, first the protein complexes with copper ions. Then, in
a second step, this protein-bound copper chelates BCA to give an intense
purple colour. The chromophore produced is more stable than the one
produced in the Lowry assay. Additional advantages include high
sensitivity (100 times higher than the Biuret), a simple commercially
available two-solution reagent, ease of procedure, and compatibility
with a large number of extraneous materials commonly found in protein
preparations. However, Smith assay development requires long incubation
times (30 minutes to 2 hours), dependence of the incubation temperature
of samples and absorbance variation with time. Also, the presence of
reducing agents, copper chelating agents, acidifiers, reducing sugars,
lipids and phospholipids in the buffer can affect the accuracy of the
results (Zaia et al., 1998).

The Bradford assay is the most commonly utilised assay for protein
determination as it is relatively easy to perform (Sapan \& Lundblad,
2015): negatively-charged Coomassie Brilliant Blue dye binds with
positively-charged proteins. The dye in solution is red (absorbs at 465
nm), but when it binds to basic amino acids in the protein, it becomes
blue (absorbs at 595 nm). The absorption in the sample can then be
compared with a standard curve. The Bradford assay has been recognised
to be as sensitive as the Lowry method, with a reported linear range
0.02-0.5 mg mL\textsuperscript{-1} (Okutucu et al., 2007) and less prone
to interferences (Zaia et al., 1998). However, it exhibited some
variations depending on the kind of protein (only detects proteins
larger than 3 kDa). Besides, detergents such as sodium dodecyl sulfate
(SDS), Triton X-100 and commercial glassware detergents, give excessive
interfering colour in the assay (Bradford, 1976), and the method has a
narrow range of linearity and poor precision (Dilena et al., 1983). A
minor disadvantage of this assay is that the dye adsorbs to glassware,
cuvettes, skin, and clothing.

Finally, the Pesce and Strande assay is based on the simultaneous mixing
of proteins and Ponceau-S dye with trichloroacetic acid (TCA), and then
the resulting precipitate is dissolved in sodium hydroxide giving a
violet colour. This assay is considered practical, capable of good
precision, and providing adequate accuracy, with a reported linear range
of 0.1-0.3 mg mL\textsuperscript{-1} (Okutucu et al., 2007). It is
recommended for routine clinical chemistry laboratories (Dilena et al.,
1983).

The importance of protein determination is, however, reason enough to
justify the investment in the development of a sensor that would allow
the determination of the concentrations of interest with minimum sample
preparation, and fast.

Recently, fluorescent sensors have caught the attention of many
scientists due to their huge advantages such as simple equipment, high
sensitivity and selectivity. There is thus a high demand for the
development of simple and stable fluorescent sensitive molecules.

Several fluorescent reagents for protein detections and quantifications
based on emission enhancement have been reported, such as NanoOrange,
fluorescamine, \emph{o} -phthaldialdehyde, cyanine dyes, and SYPRO Ruby
(Granzhan \& Ihmels, 2005; Tong et al., 2007). All these fluorophores
were used in batch analysis in solution, along with lengthy procedures,
comprising carefully timed steps, which resulted in small Stokes shifts
and nonlinear calibration curves (Tong et al., 2007). Besides, the
fluorescamine dye is unstable.

The majority of the fluorophore molecules lose their ability to emit
light when they form dimers or aggregate and therefore, can only be used
if highly diluted (Jin et al., 2011). The advantage of the fluorophore
used in this paper is that it emits when in the solid-state, which is
the main reason to use it in fluorescence probes (Jin et al., 2011).

1,4-Diketo-3,6-diarylpyrrolo{[}3,4-\emph{c} {]}pyrroles (DPP) and
derivatives represent a class of brilliant red and intense fluorescent
high-performance pigments, that gained wide attention in recent years
due to their outstanding properties, namely large extinction
coefficients, high fluorescent quantum yields, environment and heat
stability which make them excellent candidates for fluorescent sensors
(Kaur \& Choi, 2015; Zhang et al., 2018).

The --NH groups of DPP are prone to form strong hydrogen bonds with
electronegative ions like F\textsuperscript{-} or be easily
deprotonated. Therefore, an alkylated DPP derivative was synthetized to
avoid possible interferences from those ions. The presence of long alkyl
chains on both lactam N atoms, besides inhibiting the response to
fluoride ion, also favours the solubility of the DPP in organic solvents
(Qu, Hua, \& Tian, 2010).

Initially, it was envisaged that aldehyde groups in the fluorescent
molecule would be needed to assure the necessary chemical affinity with
the target proteins through their amino functional groups. Nevertheless,
a DPP derivative with ester groups was also prepared and tested, for
comparison. Figure 1 shows the molecular structure of the
DPP-dialdehyde, and DPP-diester used in this work. The interaction of
the formyl groups with proteins are expected to increase the rigidity of
the recognition molecule, leading to an increase in the fluorescence
intensity.

The measurement of proteins in human urine provides an essential tool in
the diagnosis of renal diseases. Healthy kidneys should only filter tiny
(trace) amounts of proteins into the urine as most protein molecules are
too large for the filters (glomeruli). The reference range in healthy
adult males and females is 10-150 mg L\textsuperscript{-1} or 20-140
mg/24 h (Yalamati, Karra, \& Bhongir, 2016). Higher levels indicate a
condition known as ``Proteinuria'', requiring medical follow-up.

The new sensor was tested analysing healthy people's urine samples
(detection of low protein levels), and results were compared with the
ones obtained with the highly sensitive spectrophotometric Pesce and
Strande (Ponceau-S/TCA) method.

2. Experimental

2.1 Reagents

Ponceau-S dye from Sigma-Aldrich (Madrid, Spain), trichloroacetic acid
(TCA) and sodium hydroxide from Panreac (Barcelona, Spain), and sodium
chloride from Pronolab (Lisbon, Portugal) were used. Bovine serum
albumin (BSA) was from Sigma-Aldrich and has been kept in a cold dark
place. Ultrapure water was produced by a Milli-Q system from Millipore
(Mississauga, Canada). Sodium fluoride from Fluka (Buchs, Switzerland),
sodium dodecyl sulfate from Riedel-de Haën (Seelze, Germany), and
acetone and glucose from Merck (Darmstadt, Germany) were used to test
for possible interferences.

The DPP derivatives were synthetized by a new procedure that will be
reported elsewhere(Agazzi et al., 2019). The DPP derivatives applied on
the optical fibre tip were dissolved in chloroform (Sigma-Aldrich).

2.2 Solutions and standards preparation

Ponceau-S stock solution was prepared by dissolving 1 g of Ponceau-S dye
in 25 mL of Milli-Q water. A trichloroacetic acid (TCA) solution at 300
g L\textsuperscript{-1} was prepared by diluting the appropriate volume
of TCA in Milli-Q water. The Ponceau-S/TCA concentrated reagent was
prepared by diluting 10 mL of the Ponceau-S stock solution to 500 mL
with the TCA solution.

Sodium hydroxide solution (0.8 g L\textsuperscript{-1}), sodium chloride
solution (0.9\%) and a stock solution of 1 g L\textsuperscript{-1} BSA
were prepared by dissolving the appropriate amounts in Milli-Q water.

For the Ponceau-S/TCA method, BSA standards were prepared daily in a
saline solution (0.9\% sodium chloride) from the BSA stock solution. An
equivalent sodium chloride amount was added to the urine samples
(Ballantyne, Gibbons, \& O'Reilly, 1993). For the optrode method, BSA
standards were prepared daily by diluting the BSA stock solution with
Milli-Q water.

2.3 Samples

Seven urine samples were collected from the same family (adults and
children) and analysed a few hours after collection. All samples were
previously filtered through a Whatman filter paper grade 4, pore size
20-25 \selectlanguage{greek}µ\selectlanguage{english}m (Maidstone, England) to remove coarse particles.

2.4 FTIR-ATR measurements

In order to chemically characterise and follow the interaction of the
protein with the Di-CHODPP sensing layer, infrared spectra of Di-CHODPP
before and after being in contact with BSA were acquired. An infrared
spectrometer (Bruker Alpha Platinum, Germany) equipped with a single
reflection diamond ATR module, with a resolution of 4
cm\textsuperscript{-1} and 32 scans, in the mid-infrared region
(4000-600 cm\textsuperscript{-1}) was employed. Analyses were performed
in a room with controlled temperature (23 \selectlanguage{ngerman}ºC) and humidity (35\%).

Background was obtained recording a spectrum at the ambient air. A 1 cm
x 1 cm of a clean metal plate was used as deposition surface for
FTIR-ATR measurements. A spectrum of the clean metal plate was acquired.
The metal plate was then coated with the Di-CHODPP compound dissolved in
chloroform, the solvent was left to evaporate, and the metal plate with
the Di-CHODPP was analysed. Afterwards, a drop of a 500 mg
L\textsuperscript{-1} BSA solution was placed above the Di-CHODPP
coating, left to evaporate and a new spectrum was acquired.

2.5 Coating of the optical fibre tip

The optical fibre used was a pure fused silica core of 1000 \selectlanguage{greek}µ\selectlanguage{english}m, from
Sarspec (Vila Nova de Gaia, Portugal). Before coating the fibre, the
fibre tip needed to be prepared: around 1 cm of the external buffer was
cut with a blade and removed, exposing the cladding. Then, using a
Micro-strip from Micro Electronics Inc. (Massachusetts, USA), the
cladding of the fibre was removed, exposing around to 2 to 3 mm of the
fibre core. Finally, the core was gently cleaned with ethanol before
coating. The exposed core of the fibre was then dipped into the
Di-CHODPP chloroform solution, removed, and left to dry for at least 3
hours, but preferably overnight.

2.6 Optical sensor set-up

Figure 2 shows a scheme of the experimental set-up. A light source
DH-2000 from Ocean Optics (Florida, USA) equipped with a deuterium lamp
was connected to the sensing fibre by an SMA-905 connector. The UV
excitation energy reaches the other end (the coated tip) of the fibre,
which was introduced into a PEEK home-made cell, possessing
approximately 1 mL reservoir with a filling entrance at the top, and an
exhaust aperture at the bottom (drain). The coated tip of the optical
fibre was positioned at a 90\selectlanguage{ngerman}º angle from a collimating lens, a convex
flat lens 1'', with a focal distance of 30 mm from Sarspec. The visible
light emitted from the coated tip and collected by the collimator lens
was guided to another pure fused silica fibre of 600 \selectlanguage{greek}µ\selectlanguage{english}m (core) connected
to a fluorescence spectrophotometer Scanspec from Scansci (Vila Nova de
Gaia, Portugal). The fluorescence spectrophotometer was connected to a
PC by a USB cable. The acquisition software used was Spectrascan from
Scansci.

2.7 Procedure for analysis

2.7.1 The Ponceau-S/TCA method

Total protein was quantitated following the already described procedure
for Ponceau-S/TCA method (Marshall \& Williams, 2000; Pesce \& Strande,
1973): 500.0 \selectlanguage{greek}µ\selectlanguage{english}L of Ponceau-S/TCA concentrated reagent was mixed with 5.0
mL of urine sample or BSA standard, and the mixture was centrifuged with
a centrifuge Mixtasel Mod. 540 from Selecta (Barcelona, Spain) at 3500
rpm for 20 minutes (Pesce \& Strande, 1973). After centrifugation, the
supernatant was discarded, and the precipitate was dissolved in 5.0 mL
of sodium hydroxide (0.8 g L\textsuperscript{-1}).

The absorbance of the basic solutions was measured at 560 nm with a
double beam UV-Vis spectrophotometer Shimadzu UV-2101 PC (Kyoto, Japan),
and measurements were made against Milli-Q water. Calibration line was
drawn plotting the absorbance of the BSA treated sample \emph{vs} . BSA
concentrations, and quantification of proteins in the urine sample was
obtained by entering with the absorbance corresponding to urine treated
solution in the calibration line equation.

2.7.2 The optical fibre sensor method

BSA standards were prepared from 10.0 to 60.0 mg L\textsuperscript{-1}.

Urine samples were prepared according to the following procedure: 500.0
\selectlanguage{greek}µ\selectlanguage{english}L of TCA (300 g L\textsuperscript{-1}) was added to 5.0 mL of the urine
sample. The mixture was filtered using a disposable syringe to force the
mixture through a membrane filter of 0.45 \selectlanguage{greek}µ\selectlanguage{english}m from Millipore, mounted in
an appropriated filter holder Swinnex, also from Millipore. After
filtration, the filter was removed with a plastic tweezer and carefully
washed with 4.0 mL of sodium hydroxide solution (0.8 g
L\textsuperscript{-1}) to dissolve all the proteins retained in the
filter membrane. The solution was transferred to a 5.0 ml volumetric
flask, and volume was completed with a sodium hydroxide solution using a
Pasteur pipet.

Before initiating measurements, the coated optical fibre was introduced
into the home-made liquid chamber, as represented in Figure 1, and the
light source (deuterium lamp) was left to stabilise for 30 minutes.

After light source stabilisation, measurements could be initiated. Each
BSA standard or the treated urine sample solution was poured through the
top hole of the cell with a Pasteur pipet, and the chamber top was
closed with a screw thread. The Vis spectrum (in intensity units) was
saved after stabilisation. Once the measurement was saved, the chamber
was opened, unscrewing the two screws, one at the top and another at the
bottom, and the sample was evacuated by gravity. The chamber was washed
with Milli-Q water between analysis. A calibration line was derived with
the fluorescent intensity obtained with the BSA solutions. The maximum
fluorescent intensity for each urine treated sample was entered in the
equation of the calibration line.

3. Results and discussion

3.1 Interaction between Di-CHODPP and the proteins

Experiments with the diester Di-CO\textsubscript{2}MeDPP did not show
any changes in the emission spectrum when the compound was put in
contact with BSA. This result seems to indicate that the interaction
between the protein and the Di-CHODPP does not involve the formation of
a hydrogen bond but, probably, a covalent bond involving the formyl
groups (that are much more reactive than ester groups). Because the
recognition process is very fast and reversible, that covalent bond must
be weak. Presumably, a hemiaminal bond (and not an imine bond) is formed
from the interaction of the amino groups of the protein and the formyl
groups of the recognition molecule, as illustrated in Figure 3. This
interaction is reversible, and the di-aldehyde functions are quickly
recovered after immersion of the coated fibre in water.

Figure 4 shows the IR spectrum of the Di-CHODPP before and after
contacting with BSA solution. There are two bands in the spectra of
Di-CHODPP related with C=O vibration: at 1662 cm\textsuperscript{-1} and
at 1696 cm\textsuperscript{-1}. The first one can be attributed to the
C=O stretching mode of the lactam, and, as expected, its intensity
remained unchanged after the interaction with the protein. The second
band can be attributed to the carbonyl group of the aldehyde, and its
intensity, as expected, did decrease after the interaction with the
protein.

A small shoulder at 1111cm\textsuperscript{-1} is consistent with the
formation of a C--N bond and is highly characteristic of a C--N
stretching band of a secondary amine. This band was not found in the ATR
spectra of pure BSA, as can be seen in Figure 4.

The increasing intensity of the band at \textasciitilde{}3300
cm\textsuperscript{-1}, typical of O--H and N--H bonds(Bellamy, 1975),
also corroborates the formation of the hemiaminal function.

All the listed observations are consistent with the hemiaminal formation
hypothesis, and with the reversibility of the protein interaction, very
important to assure sensor calibration and reuse.

3.2 Sensor analytical signal and calibration

After solvent evaporation, the coating (Di-CHODPP) on the tip of the
optical fibre was easily confirmed by visual inspection, due to the
presence of the orange colour of the compound. Figure 5 shows the
fluorescent emission spectra of the coated fibre in contact with Milli-Q
water and in contact with a 100 mg L\textsuperscript{-1} BSA solution.

Figure 5 shows the fluorescent emission spectrum of the coated fibre in
contact with Milli-Q water. When the sensor is put in contact with a
100.0 mg L\textsuperscript{-1} BSA solution, the intensity of the peak
at 581 nm showed a small increase, compared to the peak intensity in
water. Despite of looking like a modest increase in the fluorescent
intensity, due to the yy scale of Figure 5, the changes on the emission
intensity at 581nm were significant enough to be selected as the
analytical signal for protein quantification. The interaction between
Di-CHODPP and BSA was reversible, and intensity emission found in water
was rapidly restored after flushing the cell with Milli-Q water and
refilling it.

Figure 6 shows the emission intensity at 581 nm obtained for the BSA
standards analysed. The graph shows a linear working range between 10 mg
L\textsuperscript{-1} (LOQ) and 100 mg L\textsuperscript{-1} of BSA.

3.3 Validation of the direct BSA calibration

To evaluate the possibility of using BSA standards directly, without TCA
pre-treatment similar to the one used in the Ponceau-S/TCA method, a
60.0 mg L\textsuperscript{-1} BSA standard was analysed by the optrode,
both directly, and after pre-treatment. The pre-treatment consisted in
the TCA addition, followed by filtration and protein dissolution with
sodium hydroxide solution, as described in 2.7.2 procedure. It was found
that fluorescence emission spectra were equal, and the intensity of the
peak at 581 nm was exactly the same. These results allowed to conclude
that BSA standards could be analysed directly, without any
pre-treatment, making calibration simpler and fast than in the
Ponceau-S/TCA method.

The removal of interfering substances from urine, by protein
precipitation with TCA, and redissolution of the proteins in NaOH has
been widely used in the vast majority of methods for protein
determination (Choi et al., 1993; Noble \& Bailey, 2009; Yalamati,
Bhongir, Karra, \& Beedu, 2015). The elimination of this step would
allow saving a considerable amount of time, and although it seems to be
an unrealistic simplification, it deserves to be tested with the urine
samples. Therefore, an aliquot of urine was analysed directly and after
TCA addition, filtration and proteins dissolution with sodium hydroxide
solution.

The analysis of both aliquots, with and without TCA addition, with the
new optic fibre sensor, revealed that sample without treatment showed a
less intensity signal at 581 nm when compared with the one obtained with
the treated sample. This study allowed to conclude that urine samples
needed to be pre-treated (TCA/filtration/dissolution), like the vast
majority of methods for protein determination, to remove interfering
substances from urine that could inhibit the interaction between
proteins and the coated sensor, giving low and erroneous results.

3.4 Interference studies

Although each method for protein determination counts with particular
advantages and disadvantages, the great majority of them is prone to
interferences from numerous substances that could be found in biological
samples, such as Tris buffer, acetone, sodium dodecyl sulfate (SDS),
Triton-X, sugars, EDTA, and ethanol, among others (Bradford, 1976).

A 50.0 mg L\textsuperscript{-1} BSA standard solution was analysed using
the optical sensor, using the procedure reported in section 2.7.2 for
BSA standards, and the respective emission spectrum was recorded. To
test some of the most often reported interference compounds, solutions
containing the same amount of BSA (50.0 mg L\textsuperscript{-1}) and
the substances presented in Table 1A were prepared and analysed with the
fluorescent optical sensor.

DPP compounds were reported to be highly reactive and selective for
fluoride anions (Qu et al., 2010), due to the intermolecular proton
transfer between a hydrogen atom on the lactam N positions of the DPP
and the fluoride anion. In spite of having blocked those positions by
choosing a compound where the referred protons have been replaced by
alkyl groups, the sensor was also tested with aqueous fluoride solutions
(see Table 1B).

Fluorescence emission spectra obtained with BSA solutions with and
without the substances listed in Table 1A were compared, and no
interference was noticed with SDS, glucose and acetone, as no changes in
the BSA fluorescence intensity were observed.

The intensity of fluorescence observed with the fluoride ion solution
(Table 1B) was compared against the fluorescence spectrum in Milli-Q
water. As expected, no differences were observed between spectra,
meaning that fluoride ion did not interact with the Di-CHODPP. This
result validates the choice of N,N-dialkylated Di-CHODPP.

3.5 Analysis of urine samples by the Ponceau-S/TCA method and by the new
optrode sensor

Protein was quantified in seven urine samples using the new optrode. BSA
solutions of known concentration were used to prepare a standard
calibration line covering the range from 10.0 to 60.0 mg
L\textsuperscript{-1}. The concentration of the unknown urinary protein
samples was then determined by entering with the intensity of emission
of the TCA treated sample in the BSA calibration line. For comparison,
the protein amount in the urine samples was also measured by the
Ponceau-S/TCA spectrophotometric method. Table 2 shows the obtained
protein content found in urine samples using both methods.

The limit of detection (LOD) and the limit of quantification (LOQ) were
calculated from the calibration curve and from the standards errors
estimate S\textsubscript{yx}, that measures the variability or scatter
of the observed values around the regression line. The LOD
(concentration corresponding to a signal = intercept +
3S\textsubscript{yx}) for the optrode was 3.1 mg
L\textsuperscript{-1}while the LOQ (concentration corresponding to
signal = intercept + 10S\textsubscript{yx}) was 10.4 mg
L\textsuperscript{-1}. LOD and LOQ for the optrode method were not
significantly different from the values found for the Ponceau-S/TCA
method, 3.4 mg L\textsuperscript{-1} and 11.4 mg L\textsuperscript{-1}
for LOD and LOQ, respectively.

The values in Table 2, obtained with the two methods, were statistically
compared using a paired \emph{t-} test, and it was found that the
difference in the mean values between the results of the two methods was
not great enough to exclude the possibility that the difference was due
to random variability (P = 0.05). An \emph{F} -test was running for
comparison of standard deviations, and the Ponceau-S/TCA method was more
precise than the optical sensor method (\selectlanguage{greek}α \selectlanguage{english}= 0.05), in all cases, except
for sample 4, where standard deviations did not differ significantly (\selectlanguage{greek}α
\selectlanguage{english}= 0.05).

Healthy adults excrete less than 150 mg of total protein per 24 h
(Waller, Ward, Mahan, \& Wismatt, 1989). Considering an average volume
of urine excretion/day of 1.5 L (Dube, Girouard, Leclerc, \& Douville,
2005), the values from Table 1 correspond to total protein values
ranging from 23 to 52 mg. These are low values classified as ``negative
result'' (less than 100 mg L\textsuperscript{-1}) typically found in
healthy individuals. Results show the power of the method to quantify
low concentrations, and higher values, like the ones found in
proteinuria conditions, above 300 mg L\textsuperscript{-1}, could be
easily detected and quantified, by simply diluting the sample. The
sensor was used daily for two weeks with no loss in sensitivity.

4. Conclusions

A fluorescent optical sensor for the detection of total proteins based
on Di-CHODPP is present. The Di-CHODPP showed interesting and unusual
properties of retaining its fluorescent capability when in solid-state,
which is particularly important when used as a recognition molecule in
an optical sensor. Besides, it is insoluble in water and experienced a
fluorescence enhancement in the presence of proteins. The designed
analytical setup consisted of a UV light injected in the optical fibre
coated with the Di-CHODPP at stripped the end, which entered a home-made
cell with a 1 mL cavity with the sample. The interaction between
proteins and the coated tip of the fibre is very fast and reversible
with a simple water flush. Experiments with BSA allowed to compare the
sensor detection limit (3.1 mg L\textsuperscript{-1}) with the LOD of
other conventional methods, such as Ponceau-S/TCA spectrophotometric
method (3.4 mg L\textsuperscript{-1}).

The new fluorescent sensor was applied to the detection of the total
proteins in human urine and results were not statistically different
from the ones obtained with the Ponceau-S/TCA spectrophotometric method.
The new optical sensor, showed, however, some advantage over the
Ponceau-S/TCA traditional method, namely in the economy of reagents and
analysis time, as standards do not need to be treated with
trichloroacetic acid, nor centrifuged. The new sensor does not suffer
from interference from sodium dodecyl sulfate, acetone or glucose, the
most common interfering compounds in conventional spectrophotometric
methods. Besides, the presence of fluoride ions in solution did not
change the fluorescence intensity of the sensor. Summing up, sensor
reliability in the determination of total proteins in urine samples has
been demonstrated.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Thanks are due to FCT/MCTES for the financial support to CESAM
(UID/AMB/50017/2019), through national funds. M. Ver\selectlanguage{ngerman}íssimo is funded by
national funds (OE), through FCT - Fundação para a Ciência e a
Tecnologia, I.P., in the scope of the framework contract foreseen in the
numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of
August 29, changed by Law 57/2017, of July 19.

Thanks also to the University of Aveiro and FCT for the financial
support to project PTDC/QEQ-QOR/6160/2014 and the QOPNA research Unit
(FCT UID/QUI/00062/2019) through national funds and, where applicable,
co-financed by FEDER, within the PT2020 Partnership Agreement, and to
the Portuguese NMR Network. Vítor A.S. Almodôvar thanks FCT for his
doctoral grant (SFRH/BD/135598/2018).

References

Agazzi, M. L., Almodôvar, V. A. S., Gsponer, N. S., Bertolotti, S.,
Durantini, E. N., \& Tomé, A. C. (2019). Diketopyrrolopyrrole--fullerene
C60 architectures as highly efficient heavy atom-free photosensitizers:
synthesis, photophysical properties and photodynamic
activity.\emph{Organic \& Biomolecular Chemistry} , (Submitted),
Submitted.

Ballantyne, F. C., Gibbons, J., \& O'Reilly, D. S. J. (1993). Urine
albumin should replace total protein for the assessment of glomerular
proteinuria. \emph{Annals of Clinical Biochemistry} , \emph{30} ,
101--103. https://doi.org/10.1177/000456329303000119

Bellamy, L. J. (1975). \emph{The infra-red spectra of complex molecules}
. (C. and Hall, Ed.) (3rd ed.). London: John Wiley \& Sons.

Bradford, M. M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. \emph{Analytical Biochemistry} , \emph{72}
(1--2), 248--254. https://doi.org/10.1016/0003-2697(76)90527-3

Choi, W. S., Chung, K. J., Chang, M. S., Chun, J. K., \& Lee, H. W.
(1993). A Turbidimetric Determination of Protein by Trichloroacetic
Acid. \emph{Arch. Pharm. Res.} , \emph{16} (1), 57--61.

Dilena, B. A., Penberthy, L. A., \& Fraser, C. G. (1983). Six methods
for determining urinary protein compared. \emph{Clinical Chemistry}
,\emph{29} (3), 553--557.

Dube, J., Girouard, J., Leclerc, P., \& Douville, P. (2005). Problems
with the estimation of urine protein by automated assays. \emph{Clinical
Biochemistry} , \emph{38} (5), 479--485.
https://doi.org/10.1016/j.clinbiochem.2004.12.010

FAO. (2003). \emph{Food energy-methods of analysis and conversion
factors} . (Food and Agriculture Organization of the United Nations,
Ed.). Rome. Retrieved from
http://www.fao.org/uploads/media/FAO\_2003\_Food\_Energy\_02.pdf

Granzhan, A., \& Ihmels, H. (2005). N-aryl-9-amino-substituted
acridizinium derivatives as fluorescent ``light-up'' probes for DNA and
protein detection. \emph{Organic Letters} , \emph{7} (23), 5119--5122.
https://doi.org/10.1021/ol051624i

Jin, Y., Xu, Y., Liu, Y., Wang, L., Jiang, H., Li, X., \& Cao, D.
(2011). Synthesis of novel diketopyrrolopyrrole-based luminophores
showing crystallization-induced emission enhancement
properties.\emph{Dyes and Pigments} , \emph{90} (3), 311--318.
https://doi.org/10.1016/j.dyepig.2011.01.005

Kaur, M., \& Choi, D. H. (2015). Diketopyrrolopyrrole: Brilliant red
pigment dye-based fluorescent probes and their
applications.\emph{Chemical Society Reviews} , \emph{44} (1), 58--77.
https://doi.org/10.1039/c4cs00248b

Mæhre, H. K., Dalheim, L., Edvinsen, G. K., Elvevoll, E. O., \& Jensen,
I. (2018). Protein Determination---Method Matters. \emph{Foods}
,\emph{7} (1), 1--11. https://doi.org/10.1016/S0886-3350(13)80287-8

Marshall, T., \& Williams, K. M. (2000). Total protein determination in
urine: Elimination of a differential response between the coomassie blue
and pyrogallol red protein dye-binding assays. \emph{Clinical Chemistry}
, \emph{46} (3), 392--398.

Moore, J. C., Devries, J. W., Lipp, M., Griffiths, J. C., \& Abernethy,
D. R. (2010). Total Protein Methods and Their Potential Utility to
Reduce the Risk of Food Protein Adulteration. \emph{Comprehensive
Reviews in Food Science and Food Safety} , \emph{9} , 330--357.
https://doi.org/1oi 10.1111/j.1541-4337.2010.00114.x

Noble, J. E., \& Bailey, M. J. A. (2009). Quantitation of Protein.
In\emph{Methods in Enzymology} (Vol. 463, pp. 73--95). Elsevier Inc.
https://doi.org/10.1016/S0076-6879(09)63008-1

Okutucu, B., Dinçer, A., Habib, Ö., \& Figen, Z. (2007). Comparison of
five methods for determination of total plasma protein
concentration.\emph{J. Biochem. Biophys. Methods} , \emph{70} ,
709--711. https://doi.org/10.1016/j.jbbm.2007.05.009

Olson, B. J. S. C., \& Markwell, J. (2007). Assays for determination of
protein concentration. \emph{Current Protocols in Protein Science}
,\emph{48} (1), 3.4.1-3.4.29. https://doi.org/10.1002/cpph.3

Pesce, M. A., \& Strande, C. S. (1973). A new micromethod for
determination of protein in cerebrospinal fluid and urine.\emph{Clinical
Chemistry} , \emph{19} (11), 1265--1267.

Qu, Y., Hua, J., \& Tian, H. (2010). Colorimetric and Ratiometric Red
Fluorescent Chemosensor for Fluoride Ion Based on
Diketopyrrolopyrrole.\emph{Organic Letters} , \emph{12} (15),
3320--3323. https://doi.org/10.1039/B915340c

Sapan, C. V, \& Lundblad, R. L. (2015). Review of methods for
determination of total protein and peptide concentration in biological
samples. \emph{Proteomics Clin. Appl} , \emph{9} , 268--276.
https://doi.org/10.1002/prca.201400088

Saxena, J., Baunthiyal, M., \& Ravi, I. (2015). \emph{Laboratory Manual
of Microbiology, Biochemistry and Molecular Biology} . (Scientific
Publishers (India), Ed.). India.

Tong, H., Hong, Y., Dong, Y., Häussler, M., Li, Z., Lam, J. W. Y.,
\ldots{} Tang, B. Z. (2007). Protein detection and quantitation by
tetraphenylethene-based fluorescent probes with aggregation-induced
emission characteristics. \emph{Journal of Physical Chemistry B}
,\emph{111} (40), 11817--11823. https://doi.org/10.1021/jp073147m

Waller, K. V, Ward, K. M., Mahan, J. D., \& Wismatt, D. K. (1989).
Current concepts in proteinuria. \emph{Clinical Chemistry} ,\emph{35}
(5), 755--765.

Yalamati, P., Bhongir, A. V., Karra, M., \& Beedu, S. R. (2015).
Comparative analysis of urinary total proteins by bicinchoninic acid and
pyrogallol red molybdate methods. \emph{Journal of Clinical and
Diagnostic Research} , \emph{9} (8), BC01--BC04.
https://doi.org/10.7860/JCDR/2015/13543.6313

Yalamati, P., Karra, M. L., \& Bhongir, A. V. (2016). Comparison of
Urinary Total Proteins by Four Different Methods. \emph{Indian Journal
of Clinical Biochemistry} , \emph{31} (4), 463--467.
https://doi.org/10.1007/s12291-016-0551-3

Zaia, D. A. M., Zaia, C. T. B. V., \& Lichtig, J. (1998). Determinação
de proteínas totais via espectrofometria: Vantagens e desvantagens dos
métodos existentes. \emph{Quimica Nova} , \emph{21} (6), 787--793.
https://doi.org/10.1590/S0100-40421998000600020

Zhang, S., Sun, T., Xiao, D., Yuan, F., Li, T., Wang, E., \ldots{} Niu,
Q. (2018). A dual-responsive colorimetric and fluorescent chemosensor
based on diketopyrrolopyrrole derivative for naked-eye detection of Fe3+
and its practical application. \emph{Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy} , \emph{189} , 594--600.
https://doi.org/10.1016/j.saa.2017.09.001\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} Molecular structures of the two DPP derivatives tested
as selective membranes of the optical sensor.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} Experimental setup used with the fluorescence sensor.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} Envisaged interaction between the Di-CHODPP and the
protein amino group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} IR Spectrum of the Di-CHODPP with (grey line) and
without (black line) BSA. The light grey line represents pure BSA
spectrum.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5} Fluorescence emission spectra of the Di-CHODPP in
contact with Milli-Q water (black dots), and in contact with a 100 mg
L\textsuperscript{-1} BSA solution (gray line).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6} Intensity of fluorescence emission at 581 nm\emph{vs.}
BSA concentration (mg L\textsuperscript{-1}).

\textbf{Table 1} Substances tested for interference.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[b]{0.48\columnwidth}\raggedright\strut
\textbf{Substances in 50.0 mg L\textsuperscript{-1} BSA}\strut
\end{minipage} & \begin{minipage}[b]{0.48\columnwidth}\raggedright\strut
\textbf{Concentration}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Sodium dodecyl sulfate (SDS)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
1\% (w/v)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
D-Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
1 M\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Acetone\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
5\% (v/v)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\textbf{Substances in Milli-Q water}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\textbf{Concentration}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Fluoride ion (NaF)\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
500 mg L\textsuperscript{-1}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2} Protein content in urine samples obtained by the
optical sensor and by the Ponceau-S/TCA spectrophotometric method.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.32\columnwidth}\raggedright\strut
\textbf{Samples}\strut
\end{minipage} & \begin{minipage}[b]{0.32\columnwidth}\raggedright\strut
\textbf{Optical sensor} \textbf{method} \textbf{(mg
L\textsuperscript{-1})}\strut
\end{minipage} & \begin{minipage}[b]{0.32\columnwidth}\raggedright\strut
\textbf{Ponceau-S/TCA method} \textbf{(mg L\textsuperscript{-1})}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
23.3 \selectlanguage{ngerman}± 3.7\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
23.6 \selectlanguage{ngerman}± 2.4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
14.7 \selectlanguage{ngerman}± 4.3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
15.1 \selectlanguage{ngerman}± 2.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
11.7 \selectlanguage{ngerman}± 3.4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
12.0 \selectlanguage{ngerman}± 2.2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
26.0 \selectlanguage{ngerman}± 2.1\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
26.3 \selectlanguage{ngerman}± 2.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
15.1 \selectlanguage{ngerman}± 2.5\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
14.6 \selectlanguage{ngerman}± 2.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
23.5 \selectlanguage{ngerman}± 2.8\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
23.3 \selectlanguage{ngerman}± 2.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
14.3 \selectlanguage{ngerman}± 3.2\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
14.6 \selectlanguage{ngerman}± 2.5\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure5/Figure5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure6/Figure6}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

