Analytica Chimica Acta 1175 (2021) 338753

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Analytica Chimica Acta

journal homepage: www.elsevier.com/locate/aca

The comparison between light-scattering detectors based on LED and

photodiode for immunoprecipitation assays of transferrin and ferritin
Kamil Strzelak *, Agnieszka Czajkowska , Robert Koncki
University of Warsaw, Faculty of Chemistry, Pasteura 1, 02-093, Warsaw, Poland

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Detectors based on LED and photo-

diode (PD) as light sensors have been
compared and examined for
immunoprecipitation.

Multicommutated flow system with
light-scattering detectors were opti-
mized for detection of transferrin and
ferritin.

Both detectors enabled the determi-
nation of transferrin but only LED-PD
were efficient for detection of serum
ferritin.

a r t i c l e i n f o a b s t r a c t

Article history: Undoubtedly, light-emitting diodes (LEDs) and photodiodes (PDs) are indispensable optoelectronic de-
Received 21 March 2021 vices in modern analytical chemistry. LEDs can serve as either light emitters or detectors, thus being an
Received in revised form alternative to the most popular detection systems consisted of PD. In this contribution, a comparison
12 May 2021
between LED-LED and LED-PD detectors, operating in turbidimetric and nephelometric modes, has been
Accepted 10 June 2021
carried out for immunoprecipitation detection of transferrin and ferritin. The greatest emphasis was
Available online 11 June 2021
placed on the study of detectors responses under different measurement conditions including current
powering an emitter, amplification gain in the case of PD as detector or the construction of detection cells
Keywords:
Transferrin
designed for the Multicommutated Flow Analysis (MCFA). The assumption was to obtain the fully-
Ferritin mechanized system with simple but efficient detection system to enable the determination of iron-
Immunoprecipitation binding proteins occurring at different concentration ranges in human body. As a result, the optimized
Optoelectronics arrangements of LED-LED and LED-PD setups were characterized by similar analytical characteristics,
Flow analysis enabling the determination of transferrin with the detection limit (LOD) of 0.2 mg/L and RSDs of 2.8
e4.8% for LED-LED, and LOD of 0.1 mg/L and RSDs of 0.9e3.6% for LED-PD. In the case of ferritin
detection, only the response of the LED-PD detector was statistically distinguishable in the range of 130
e198 mg/L of protein with recorded analytical signal change of 20 mV value. The addition of polymer for
signal enhancement provided the increase of response range to 107e253 mg/L, enabling the developed
system for detection of pathological serum ferritin levels.
© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Transferrin and ferritin are well-known proteins involved in iron

* Corresponding author. homeostasis, providing iron distribution and storage throughout
E-mail address: kamil.strzelak@chem.uw.edu.pl (K. Strzelak).

https://doi.org/10.1016/j.aca.2021.338753
0003-2670/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
K. Strzelak, A. Czajkowska and R. Koncki
the body, respectively [1,2]. These proteins as well as corresponding
clinical parameters (serum iron, unsaturated and total iron binding
capacity) are considered as biological markers for assessment of
iron deficiency, which is the most widespread nutritional disorder
in the world (according to World Health Organization). Further-
more, the determination of transferrin can be useful in the moni-
toring of anemia, iron overload, chronic liver diseases, malnutrition,
chronic inflammatory disorders and malignancies [1]. It also seems
to be one of the tools allowing to study the etiology of Alzheimer's
and Parkinson's diseases [3,4] or to monitor the therapeutic ap-
plications of transferrin as a targeted drug delivery unit [5]. On the
other hand, the serum ferritin level can indicate inflammatory
diseases, cancers and chronic anemia (such as thalassemia) [1,6,7].
Such a great versatility and usefulness of these biomarkers prove
not only its contemporary clinical significance but also the future
therapeutical potential. Therefore, it is not surprising that the
proposals of novel assays for transferrin and ferritin estimations
appear in the literature constantly. Many of contributions that have
been published lately are characterized by very low limit of quan-
titation (LOQ) and relatively high sensitivity. The improvement of
these parameters has different coherence for both proteins deter-
mination, related to different reference ranges in human serum. For
transferrin determination (reference range of 1.2e3.6 g/L [1]), the
decrease of LOQ means dilution of a sample 1000-fold and more,
whilst the methods for ferritin determination (reference range of
10e250 mg/L [1]) still feature the LOQ on the level of mg/L.
The methods, developed in recent years for transferrin deter-
mination, include immunosensors based on magnetic particles [8],
SPR with boronic acid monolayer [9] or in the form of biosensor
[10] as well as fluorometric detection with magnetic molecularly
imprinted nanoparticles [11]. The publication devoted to ICP-MS
with isotope dilution touches upon a little bit different aspect of
transferrin measurements, focusing on accuracy and precision of
absolute transferrin determination instead of lowering the LOQ
parameter to incredibly low levels (still remaining below the
reference range of transferrin in human serum) [12]. In the case of
ferritin detection, the recent achievements represent immunolog-
ical methods that include immunosensors based on quantum dots
[13] or magnetic nanoparticles/chitosan composite film [14] as well
as paper-based electrochemical immunosensor [15], nano-
conjugates for photometric and florimetric detection [16] and
western blotting based on quantum dots [17]. All of above-
mentioned methods are useful to determine effectively particular
proteins in human serum but it does not mean that they can be
easily adapted for routine clinical analysis. The majority of them are
based on complex analytical multistep procedures that affect time
of analysis, precision of measurements and make difficult to
mechanize such a methodology in the form of analytical systems
for everyday practice [18].
The other approach enables the whole analytical procedure to
be relatively easy to carried out (single- or two-steps methodology)
using cost-effective analytical devices (detectors, actuators, valves)
in the combination with well-known analytical methods. Recently,
the MultiCommutated Flow Analysis (MCFA) systems for trans-
ferrin determination applying two different methodologies have
been reported [19,20]. The first one was based on photometric
detection of iron ions using ferrozine under two different mea-
surements conditions, which lead to the estimation of iron/trans-
ferrin clinical parameters and in turn to indirect determination of
transferrin [19]. The other approach applied immunonephelo-
metric method for assessment of transferrin molecules level [20].
The common part of both projects was the use of very simple op-
toelectronic detectors with light-emitting diodes (LEDs) as a light
emitters paired with another LED playing the role of radiation de-
tector. The combination between the concept of LED-LED detectors
2
Analytica Chimica Acta 1175 (2021) 338753
and flow analysis systems turned out to be sufficient for analysis of
human serum showing the good agreement with reference results
of transferrin determination. Moreover, although these systems
offered much lower sensitivity and higher LOQ than methods in the
paragraph above, the measurements were characterized by
reasonable analysis time and throughput, low complexity of prep-
aration step and analytical procedure as well as relatively high
mechanization degree (with the ability to remote control the sys-
tem using a mobile phone [20]).
Undoubtedly, LEDs are indispensable optoelectronic devices in
modern analytical chemistry. They serve as an effective source of
light with relatively narrow spectral bandwidth. Such a quasi-
monochromaticity makes LEDs suitable for most of analytical ap-
plications without additional optical filters or monochromators
[21]. Along with other features such as robustness, long lifetime,
low power consumption and mechanical durability, their use is not
limited only to batch measurements, playing a significant part in
flow analysis, chromatography, capillary electrophoresis as well as
portable sensing systems [21e23]. Under all these conditions, LEDs
can be applied for all kinds of optical detection systems including
photometric [24e26], fluorometric [23,24,27] and light-scattering
methods [20,28,29]. Moreover, the concept of using selected LEDs
instead of photodiodes to detect light has been proposed [30e32].
Nevertheless, LED-LED detectors are definitely a less popular
alternative to miniaturized optoelectronic detectors consisted of
LED as a light emitter and photodiode (PD) as a light detector.
Although there are many contributions devoted to LEDs (mostly as
a light source) and PDs in analytical chemistry, only a few studies
have compared the performance of them as detectors [21,22,33].
The conclusions of these contributions have indicated that both
optoelectronic detectors can produce adequate results but the PD
tends to exhibit better signal stability and reproducibility.
In this contribution, a comparison between LED and photodiode
in its most popular setups as light-scattering detectors has been
carried out for immunoprecipitation measurements under flow
conditions. The performance of above-mentioned optoelectronics
were tested using different constructions of flow-through cells for
both turbidimetric and nephelometric immunochemical detection
of two kinds of iron-binding proteins: transferrin and ferritin,
which are present in human serum in different ranges of concen-
tration. In the literature there is no explanation or guidance on how
LED-LED and LED-PD affect the performance of analytical method
in terms of basic analytical parameters such as limit of detection,
linearity or sensitivity. Thus, the usefulness of both detectors were
checked for the determination of high- and low-concentrated
proteins, so under the conditions of high and low turbidity,
respectively.
2. Experimental
2.1. Chemicals
For stationary measurements, albumin from human serum
(A1653, 96%) with 5-sulfosalicylic acid (abbreviated as SSA)
(S2130, 99%) were used. The immunoprecipitation detection un-
der flow analysis conditions was performed using both human
transferrin (T3705, 95%) with anti-transferrin antibody (T2027,
4.7 mg/mL) and ferritin from human spleen (F6879, 10 mg/mL) with
anti-ferritin antibody (F5012). For presented experiments, the
dilution factor of the antibody solutions for transferrin and ferritin
measurements were 5.2 and 1.5, respectively. As a potentiator used
for polymeric enhancement, poly(ethylene glycol) (PEG, 81255)
was added into an antibody solution. All of the above-mentioned
reagents were purchased from Merck (Germany). For all experi-
ments, doubly distilled water was used throughout. For the dilution
K. Strzelak, A. Czajkowska and R. Koncki Analytica Chimica Acta 1175 (2021) 338753

of antisera, 10 mmol/L phosphate buffer (pH 7.4) was used (Avantor consisted in direct mixing of protein solution with SSA directly
Performance Materials, Poland). inside the disposable acrylic cuvettes (Sarstedt, Germany), which
were placed inside the 3D printed cuvette holder. The holder
enabled the optical devices to be mounted in the position where
2.2. Instrumentations e construction and operation
diodes are opposite to each other in the turbidimetric or nephe-
lometric mode (Fig. 1A and B). The preparation of flow-through
In general, this contribution is devoted to the comparison of
detectors was carried out by material processing of polyether
optoelectronics sensing devices (LED and PD) and the evaluation of
ether ketone (PEEK, Plastics Group, Poland) using milling machine
their performance as simple, cost-effective light-scattering de-
and lathe. This polymer is easily machinable and used to produce
tectors for complex clinical analysis purpose (immunochemical
parts of high-quality, so no additional processing (like polishing) is
detection of transferrin and ferritin). During the studies, a great
needed. For turbidimetric measurements, two constructions of
attention was paid to the comparison of the detectors construction
detection cells have been proposed. The first one was characterized
and the application of basic optoelectronic devices as detection
by 10 mm optical path length with 3 mm channel diameter and the
systems under flow analysis conditions. Thus, it was indispensable
position of inlet and outlet on opposite sides of the optical path,
to prepare or set up required instrumentations which are presented
near acrylic windows separating diodes from the solution (Fig. 1C).
schematically in Fig. 1.
In Fig. 1D, the other cell design is presented with optical path length
The procedure for measurements under stationary conditions

Fig. 1. Scheme presenting the instrumentation (detectors, electronic circuits, flow system) used during the research. A: typical cuvette holder for turbidimetric stationary mea-
surements. B: typical cuvette holder for nephelometric stationary measurements. C: flow-through turbidimetric cell with 10 mm of optical path with diameter of 3 mm. D: flow-
through turbidimetric cell with 3.5 mm of optical path with diameter of 5 mm. E: flow-through nephelometric cell with the same dimensions as in D with acrylic light guides
providing the illumination of a solution by four LEDs-emitters (630 nm). F: electronic circuit for the LED-emitter (630 nm) with Arduino Mega as a current source G: setup of a
voltage measurement generated on LED-LED with the use of multimeter H: electronic circuit of photodiode transimpedance amplifier (TIA) coupled with 16 bit ADC and Arduino
Mega as a signal recorder. I: MCFA system for immunoprecipitation measurements.

3
K. Strzelak, A. Czajkowska and R. Koncki
of 3.5 mm, diameter of 5 and inlet and outlet placed one above the
other in one plane. Such a geometry was also used for arranging the
nephelometric detector (Fig. 1E). In this case, the four LEDs-
emitters were attached (using LEGO bricks as a diode holders
with drilled hole on the top) to the poly(methyl methacrylate)
(PMMA) light guides with 3 mm diameter (1216.1003, Mentor,
Germany) [20]. The inner volume of all presented flow-through
cells were ca. 70 mL.
The detection systems used throughout the research included
LEDs as emitters (630 nm, OSHR5161A-NO) supplied by the Ardu-
ino Mega (Arduino, Italy) (Fig. 1F). Such a wavelength for emitter
was chosen due to the possible absorption of a radiation by sam-
ples. In the visible range, the physiological fluids exhibit significant
absorption from 380 nm to 550 nm, which corresponds to the light
absorbed by yellow-orange solutions. The longer wavelength of
LED-emitter reduces significantly such kind of spectral interfer-
ence. Depending on the approach, LED (650 nm, L-53SRC-F) or
silicone PIN photodiode (SFH 203) have been used as a light de-
tector. In the case of registering light intensity with the use of LED,
the UT70B multimeter (UNI-T, China) connected with data storage
computer via RS232 interface was applied to measure the voltage
between electrodes of LED-detector (Fig. 1G) [32]. Otherwise, the
transimpedance amplifier (TIA) (based on LM358P operational
amplifier, Texas Instruments, USA), external 16 bit analog-digital
converter (ADC) (ADS 1115, Texas Instruments, USA) and Arduino
MEGA (Fig. 1H) were used to convert, amplify and finally register
the voltage signal proportional to the light intensity detected by a
photodiode. The full-scale range of the ADC scaling was set to
2.048 V (providing the resolution of 0.0625 mV). All of above-
mentioned optoelectronics and electronic devices were pur-
chased from TME (Poland).
The Multicommutated Flow Analysis (MCFA) responsible for
immunoprecipitation measurements is presented in Fig. 1I.
Depicted arrangement of solenoid pumps (120SP12120-5TV) and
solenoid valves (100T3MP12-62-5), which were bought from Bio-
Chem Fluidics (USA), has been already presented elsewhere
[20,34,35] and it can be considered as an efficient tool allowing the
multi-tasking immunochemical procedure to perform. In short, the
four main steps were needed to be carried out to accomplish one
measurement cycle. The loading of antibody solution was con-
ducted by the actuation of pump P2 with valve V1 switched into NC
position. Whereas, the injection of antigen solution was performed
by the actuation of pump P4 and valve V2 with valve V4 switched
into NC position. The operation of both these particular devices in
relation to each other ensured the dilution of a sample up to 10-
fold, which is crucial to conduct the immunoprecipitation mea-
surements [34]. After the injection, both segments were trans-
ported to reach the detector cell by actuation of pumps P1 and P3
with energized valve V3. Subsequently, the flow was stopped and
the signal recording took place. After 180 s (or 600 s in the case of
ferritin measurements), the previous step was continued as a
cleaning one, preparing the system for next measurement cycle. All
the solenoid devices were controlled using Arduino Mega with ULN
2803 integrated circuit, powered by 1 A AC/DC adapter with 12 V
output (TME, Poland).
3. Results and discussion
3.1. Light emitting diode and photodiode as light detectors
As it has been already mentioned, two optoelectronic detection
setups based on LED or PD as light sensing devices were taken into
account. The first configuration, where LEDs played the role of both
emitter and detector (LED-LED), was assembled with low input
impedance multimeter in photovoltaic mode. Such an arrangement
4
Analytica Chimica Acta 1175 (2021) 338753
is characterized by better sensitivity for light intensity detection in
the comparison with high input impedance devices like pH-meters
[32]. There is also the significant consequence of such an approach
which is the visible sigmoidal signal response. This effect in turn
results in the narrowing of working range as well as the occurrence
of quasi-linear character of dynamic range [32,33]. Nonetheless,
LED-LED constructions have been widely applied in the case of
portable analytical platforms and flow analysis systems, being
suitable for clinical [20,24,35], environmental [36] and pharma-
ceutical [37] analysis.
Due to the fact that LED-LED detector presents much higher
sensitivity in photovoltaic mode than the LED-PD [33], the PD was
connected to the transimpedance amplifier (TIA) to balance the
difference between the devices (Fig. 1H). Such a LED-PD configu-
ration provided constant 0 V across the optoelectronic device
(photovoltaic mode). It also kept the dark current at minimum level
(the elimination of dark current is impossible due to the input offset
voltage of op-amp). TIA converted current signal generated by the
photodiode to a measurable voltage, whose value depended pro-
portionally on feedback resistor value. Subsequently, the output
signal went directly to the analog-digital converter (ADC) and then
was recorded by Arduino Mega. The application of ADC enabled the
resolution of detection to increase from 4.8 mV (10 bits for Arduino
Mega) to 0.0625 mV (16 bits of ADC for single-ended input). As a
result, the obtained signal is proportional to light intensity and thus
to transmittance, which is not proportional to the concentration
according to Lambert-Beer law for photometric/turbidimetric
measurements. There were a few reasons that can explain such a
choice under presented conditions for light-scattering detection.
Firstly, the dependence between transmittance and absorbance/
turbidance has a linear character at low absorbance values (0.3,
which corresponds to 50% of transmittance) [33,38] (please see
results hereinafter). Furthermore, the same LED-PD detection sys-
tem was applied for nephelometric measurements, where the
scattered light intensity is directly proportional to the antigen
concentration. Thus, in both cases of turbidimetry and nephelom-
etry, the photodiode signal-concentration relationship exhibited
linearity for concentrations of proteins where the transmittance
does not exceed 50% (as shown below, this is a sufficient range for
protein determination). The result is signal-to-concentration pro-
portionality for both detection modes, which makes the photo-
diode with transimpedance amplifier a very universal detector for
immunoprecipitation detection.
The discussion of performance and comparison between
particular light-scattering detectors (LED-LED and LED-PD) were
based on two precipitation assays. The first one included model
precipitation reaction between human albumin and SSA, causing
the turbidity which is proportional to the protein concentration. In
the literature, such a method (so-called the Exton method) is used
for the determination of relatively high protein concentrations
present in clinical samples like blood serum [28] and cerebrospinal
fluid [39]. The second type of assays concerned immunological
interaction between antigen and specific antibody. It leads to the
formation of immune complexes, manifesting itself as a turbidity at
some ratio of antigen and antibody concentrations. The graphical
representation of immunoprecipitation reaction is a bell-shaped
curve called Heidelberger curve, presenting the nonlinear depen-
dence between signal response and antigen concentration
[1,35,40]. The exemplary calibration curve for immunoprecipitation
detection of transferrin under flow analysis conditions (described
in chapter 3.2) is depicted in Figure S1 in the supplementary ma-
terial. The consequence of such a dependence is the possibility to
assign two different values of the determined protein concentra-
tions for a single analytical signal value. Therefore, the immuno-
precipitation measurements require special analytical procedure to
K. Strzelak, A. Czajkowska and R. Koncki
be carried out, which is presented in details elsewhere [34]. In brief,
such a procedure is based on series of dilutions with simultaneous
tracking the changes in analytical signals. It enables the correct
value of antigen concentration to be determined.
The first step to evaluate the performance of above-mentioned
simple light-scattering detectors was to carry out the cuvette
measurements. It allowed the initial conditions for further mea-
surements to be set up along with the exclusion of some variables
such as geometry of detection cell, transient-state kinetic signal or
general flow analysis character of measurements (which are
demonstrated further in this paper). The procedure was based on
precipitation assay between albumin and 10% (v/w) SSA that were
mixed with each other in the 1:1 ratio. After 20 min, the signal
values were registered serving to create many calibration curves in
the range of 0e300 mg/L protein concentrations (top of Fig. 2). It
should be emphasized that this chapter is devoted only to turbi-
dimetric detection, while the nephelometry is not show here
because of very poor results in stationary mode (signals haven't
exceeded 50 mV with the single emitter mounted perpendicular to
detector). Moreover, the conditions of nephelometric measure-
ments have been recently discussed elsewhere [20] with the use of
specially prepared flow cell (Fig. 1E).
The turbidimetric experiments were conducted to determine
the initial working parameters for further flow analysis in-
vestigations. It consisted in the monitoring of changes in detectors
Fig. 2. Top: Effect of current supplying LED-emitter on detector response during
precipitation reaction between human albumin and 5-sulfosalicylic acid. Graphs show
both direct signal, understood as a detected signal value (left) and analytical signal
which is a direct signal reduced by the signal for a background (right). The resistor of
transimpedance amplifier has been set to 150 kU. Bottom: Effect of the tran-
simpedance amplifier gain (set by the resistor) on analytical signal for three current
values (8.2, 11.7 and 15.1 mA).
5
Analytica Chimica Acta 1175 (2021) 338753
response with the change of protein concentration while the
different current supplied the LED-emitter. The top of Fig. 2 pre-
sents two signal dependencies as a function of protein concentra-
tion, involving either direct signal, understood as a signal recorded
by multimeter, or analytical signal which was calculated as a dif-
ference between obtained signal and baseline for particular con-
ditions. There are some interesting effects that can be observed. The
LED-LED-based device showed higher spectral sensitivity than the
LED-PD configuration. The optimal supplying current (1 mA) for
LED-LED was definitely insufficient for LED-PD, causing a signifi-
cant difference in calibration curve slopes (ca. 3.3 V$L/g for LED-LED
and 0.2 V$L/g for LED-PD). The saturation of a LED-LED signal
occurred for 4.7 mA current whereas the optimal value for LED-PD
was at 11.7 mA current resulting in similar calibration curve slopes
as it was in the case of 1.0 mA for LED supplying current. But as it
was mentioned before, the LED-LED response was quasi-linear and,
what is truly undesirable, it presented sensitivity close to zero for
low concentrations (for 5.0 and 10.0 mg/L under optimal condi-
tions). On the other hand, the PD signal response had a linear
character for the presented current values in the whole range of
concentrations, except the situation of detector saturation (voltage
reach the maximum scale range for ADC) for low protein concen-
trations in the case of 15.1 mA. The only limitation could be the lack
of linear relationship between generated signal and concentration
but the presented results were performed for turbidance less than
0.3 providing the linear nature of response.
In the bottom of Fig. 2, the juxtaposition of analytical signal with
protein concentration in the dependence of gain and current sup-
plying LED-emitter of LED-PD is depicted. It shows that these var-
iables have an influence on analytical parameters but their nature is
completely different. The light intensity illuminating a detector,
that directly defines the baseline signal, affects the sensitivity and
working range by increasing their values with higher currents.
More importantly, although the working range becomes wider with
the increase of current supplying an emitter, it does not apply to the
other crucial parameter which is limit of detection, LOD. For 150 kU
(marked in bottom Fig. 2 as blue lines), the lowest LOD appeared for
11.7 mA current, becoming a result of sensitivity and saturation of
specific detection system. For higher current, the limiting factor,
that dramatically increased the LOD, was the scale range of ADC
causing the saturation of detector at 15.1 mA current - the gener-
ated voltage on photodiode exceeded 2.048 V. An alternative would
be to change the ADC scale to 4.096 V, that would adversely affect
the resolution of measurements from 0.0625 mV to 0.125 mV.
For further flow analysis investigations, it has been assumed
that the current supplying LED-emitters would be selected to
achieve the baseline according to optimal stationary measure-
ments. It was ca. 1050 mV and 1800 mV for LED-LED detector and
LED-PD detector with 150 kU resistor in amplifying circuit,
respectively.
3.2. Flow analysis setup e transferrin and ferritin detection
In this section, the optical detection performance of LED-LED
and LED-PD detectors are compared with each other under flow
analysis conditions. For this purpose, the two light-scattering
methods were applied for immunoprecipitation detection of
transferrin and ferritin. Both nephelometric and turbidimetric
measurements were performed with the use of different flow cells
depicted in Fig. 1C, D and E. The results presented in Fig. 3 are
organized with three rows, where each row concerns different
setup of detector construction with corresponding detection mode
and the columns show two forms of results presentation e as an
analytical signal (difference between signal height and baseline)
and as a normalized signal (for the comparison of the relative
K. Strzelak, A. Czajkowska and R. Koncki Analytica Chimica Acta 1175 (2021) 338753

Fig. 3. Response for LED-LED and LED-PD detectors obtained during transferrin immunoassay, including three constructions of detectors (details in section 2.2). The linear fit
represents theoretical linear range starting with the LOQ value of corresponding method.

response changes between given approaches). All investigations
were conducted in the range of transferrin concentration between
0 and 50 mg/L, focusing on the antibody excess zone of calibration
curve that can be treated as a linear section of nonlinear depen-
dence (Fig. S1). Based on obtained results some crucial analytical
parameters as sensitivity, LOD (mean þ 3SD, n ¼ 3) and LOQ
(mean þ 10SD, n ¼ 3) were estimated and gathered together in
Table 1. In the case of immunochemical measurements, such pa-
rameters strongly depend not only on detection system or addi-
tional reaction enhancements but also on the primary antigen
affinity affecting the strength of the antibody-antigen interaction. It
means that even the similar investigations may vary significantly
with the use of slightly different antibody solution (even if the
concentration and antigen-antibody ratio remains the same [41]).
The first row of Fig. 3 presents the analytical signals and
normalized signals for nephelometric detection, where LED-
emitters (6 mA supply current) and the sensing device were
located perpendicular to each other (Fig. 1E). As can be seen from
Table 1, the LED-PD detector turned out to be less sensitive with
narrower linear range than LED-LED but in the same time it pro-
vided better LOD and LOQ due to very low and stable baseline. In
turn, the turbidimetric measurements with the use of flow cell with
the same dimensions as before (Fig. 1D) were characterized by
worse LOD and LOQ parameters for LED-PD but with much better
sensitivity and relatively similar values for LED-LED.
The interesting issue was the appearance of two linearity ranges
as a part of one calibration plot. It was most likely related to the
geometry of the cell, which allowed the reaction zone with higher
protein concentration to disperse or the antigen-antibody com-
plexes to begin to settle (so to leave the optical path) over time of
measurement. The last setup concerned turbidimetry with the use
of flow-through cell with 10 mm optical path (Fig. 1C). In this case,
the measurements were more sensitive than for the other designs,
however, the parameters for LED-LED did not especially improve in
the comparison to the parameters calculated for other designs (see
Table 1). Nonetheless, the smaller difference between these two
values suggests the better precision of measurements (both of them
strongly depend on the signal deviations). On the other hand, the
LED-PD response was characterized by the highest sensitivity and
the lowest LOQ value in relation to the devices presented so far.
Because this setup turned out to be the most promising, additional
investigations were conducted with 4% (v/v) PEG as a potentiator to

Table 1
The analytical parameters referring to the analytical response of different detectors, presented in Fig. 3. LOD e limit of detection, linear range - the range between limit of
quantitation (LOQ) and limit of linearity, R2 - coefficient of determination. LOD and LOQ were calculated as the mean of the blank sample plus 3 and 10 standard deviations
(n ¼ 3), respectively.

Sensitivity [V$L/g] LOD [mg/L] Linear range [mg/L] R2

Nephelometric cell 3.5 mm optical path Nephelometry LED-LED 0.65 8.0 14.2e50.0 0.990
LED-PD 0.54 2.8 7.2e30.0 0.999
Nephelometric cell 3.5 mm optical path Turbidimetry LED-LED 0.70 7.8 16.0e30.0 (1st range) 0.999
LED-PD 1.61 3.7 11.6e20.0 (1st range) 0.996
Turbidimetric cell 10 mm optical path Turbidimetry LED-LED 2.40 11.5 14.4e50.0 0.985
LED-PD 5.22 5.0 5.6e50.0 0.999
LED-LED With polymer enhancement 4.13 0.2 1.4e40.0 0.993
LED-PD With polymer enhancement 8.74 0.1 1.6e30.0 0.999

6
K. Strzelak, A. Czajkowska and R. Koncki Analytica Chimica Acta 1175 (2021) 338753

improve the determination efficiency (bottom row of Fig. 3). Such a

non-ionic polymer does not interact with protein itself but it ste-
rically excludes antigen and antibody from the part of the solvent
affecting the solubility limit of proteins [42]. Thus, PEG can be
treated as an immune aggregation enhancer that increases the
light-scattering signal which directly translates into the sensitivity
of a method [43,44]. In presented case, the enhancement was so
significant that not only the PD but also LED detection had a linear
character of a response with very low LOD and LOQ and high
sensitivity.
Some general conclusions can be drawn from Fig. 3 and above-
mentioned results. In each case of presented setups, PD-based
detection was characterized by lower LOD and LOQ parameters
than in the corresponding arrangements with LED. As it was
mentioned before, the LED-LED detectors have the sigmoidal
response, which manifests itself in lower sensitivity for low protein
concentrations. The shapes of analytical response in a form of
normalized signals in a function of time are depicted in Fig. S2 in
the supplementary material. As it was mentioned above, PEG pro-
motes the interaction between antigen and antibody increasing
also the reaction rate of immune complex formation [45]. It can be
observed in Fig. S2 as a difference between kinetics with and
without the potentiator. Nonetheless, for all turbidimetric results,
the slope of linear fit line is always higher for photodiode detector,
which can be explained due to the much bigger radiant sensitive
Fig. 4. The response of photodiode-based detector for ferritin immunoassay without
area (1 mm2) in the comparison to LED (commonly
(blue) and with (red) PEG enhancement, covering antibody excess zone and equiva-
0.25  0.25 mm) that has optimized construction to emit light. lence zone of Heidelberger curve for ferritin-antibody interactions. Depicted inset
Despite the comparisons made, it can be noticed that both presents the derivative of presented fitting lines, designating approximate working
detection systems in any configuration could be applied to deter- ranges of 130e198 mg/L for measurement without PEG and 107e253 mg/L for mea-
mine the transferrin concentration with dilution of a serum sample surements with PEG. (For interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of this article.)
at least 50-fold. But the selection of the most efficient detector can
be translated into better precision and lower sample and reagents
consumption. For the best configurations, which were turbidi- this particular case of very low-concentrated protein detection, the
metric setups in 10 mm flow through-cell, the comparison between discrepancies in analytical usefulness between LED and photodiode
LED-LED and LED-PD as sensing devices have been performed. as detectors truly appears.
Regardless of whether the chemical enhancement was provided or
not, the results were consistent with each other with relative
standard deviations (RSDs) in the range of 0.9e3.6% and 2.8e4.8% 4. Conclusions
for LED-PD and LED-LED, respectively.
The same detectors were tested for the detection of ferritin, a Hereinabove, the performance evaluation of LED and PD as a
protein found in human serum at the mg/L levels. The use of simple light detector for turbidimetric and nephelometric measurements
light-scattering detectors turned out to be partially useful for at has been carried out. For the comparison, two simple detection
least semi-quantitative analysis. The detection of ferritin immune systems were taken into account allowing both the devices to
complexes highlights the differences in already discussed behavior operate in photovoltaic mode. Such detection constructions were
of optoelectronics devices. As a result, the LED-LED detector could incorporated into three different flow-through detector designs
not detect the ferritin in the range of 0e350 mg/L, showing no signal and then examined for the detection of transferrin and ferritin
response during measurements even for 10 min of incubation time. immune complexes. In the case of transferrin determination, the
It is a consequence of very poor sensitivity of LED-LED detector for PD-based detector showed better analytical parameters like LOD,
low turbidity samples due to its sigmoidal response. Based on the LOQ and sensitivity. However, due to the fact that transferrin is a
results presented in Fig. 3, the turbidity of a sample that gives protein with a relatively high concentration and the sample must
around 18 mV of analytical signal without potentiator for LED-PD be at least 50-fold diluted, these parameters is not truly significant.
(which is a signal obtained during ferritin detection) corresponds Definitely, more important is the precision that directly influences
to 2 mV for LED-LED response in low turbidity region. Taking into errors during the estimations of analyte concentration. In this case
account that standard deviation for blank is a little bit less than also the results obtained by LED-PD detectors were slightly better
1 mV, such a signal of 2 mV is indistinguishable from the baseline (RSDs of 0.9e3.6%) but LED-LED ensured a reasonable and com-
signal. parable level (RSDs of 2.8e4.8%), enabling the transferrin deter-
Fortunately, as can be seen in Fig. 4, the response of a LED-PD mination in both cases. The situation dramatically changes in the
was statistically distinguishable in the range of 130e198 mg/L. case of detection of significantly lower concentrations of immu-
Additional polymer enhancement provided the increase of nocomplexes where detector has to respond to minor changes of
response range to 107e253 mg/L. Such a range covers the concen- scattered light. LED-LED detector was unable to distinguish any
trations assigned to upper limits of reference ranges for both adult signal for different ferritin concentration, while PD-based detector
women (10e120 mg/L) and men (20e250 mg/L) [1]. Thus, it allows was able to monitor the changes of around 20 mV. It is due to the
the presented system to be used as a pathological levels sensor of sigmoidal characteristic of LED-LED arrangement, which does not
human serum ferritin, without possibility of fully quantitative provide sufficient sensitivity towards low concentrations of an
determination of this trace analyte under presented conditions. In analyte. It proves that although LED-LED detection can be a
7
K. Strzelak, A. Czajkowska and R. Koncki
competitive alternative LED-PD as a light-scattering detector for
analysis of high-concentrated proteins, it cannot boast of sufficient
analytical parameters in the case of slightly turbid solutions.
Moreover, to the best of Authors’ knowledge presented system with
LED-PD detector is the first multicommutated flow system for
detection of ferritin in the range significant for bioanalysis.
CRediT authorship contribution statement
Kamil Strzelak: Conceptualization, Investigation, Methodology,
Software, Formal analysis, Writing e original draft, Visualization,
Funding acquisition. Agnieszka Czajkowska: Investigation, Formal
analysis. Robert Koncki: Supervision, Writing e review & editing.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
The cuvette holders presented in section 2.2 were kindly pre-
pared by Michał Michalec (Faculty of Chemistry, University of
Warsaw). These investigations were supported by the Polish Na-
tional Science Centre (Project SONATA NCN no. 2016/21/D/ST4/
00924).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.aca.2021.338753.
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