Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing
Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing
Tuomas Näreoja 0 1 2 3
Jessica M. Rosenholm 0 1 2 3
Urpo Lamminmäki 0 1 2 3
Pekka E. Hänninen 0 1 2 3
0 Pharmaceutical Sciences Laboratory, Faculty of science and engineering, Åbo akademi University , Tykistökatu 6A, 20520 Turku , Finland
1 Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet , F46 , Karolinska Universitetssjukhuset, Huddinge , 141 86 Stockholm , Sweden
2 Laboratory of Biophysics, Institute of Biomedicine and Medicity research laboratories, University of Turku , Tykistökatu 6A, 20520 Turku , Finland
3 Department of Biochemistry/Biotechnology, University of Turku , Vatselankatu 2, 20500 Turku , Finland
Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L−1 in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates.
Sandwich-type immunoassay; Time-resolved fluoroimmunoassay; Nanoparticle bioconjugate; Immunoassay interference; Nanoparticle protein corona
Eu-doped nanoparticles can be manufactured to afford high
colloidal stability; good protection of Eu-chelates from
solvent; very low self-quenching owing to long Stoke’s shift of
the Eu-chelates; density that is close to water’s density to
reduce sedimentation; and a dense coating of carboxyl groups
on their surface for efficient bioconjugation . Most
importantly the Eu-doped nanoparticles facilitate sensitive
timeresolved low background detection of Eu-emission and are
thus perfect label candidates for assays that require high
sensitivity. However, while the nanoparticle-based assay concepts
have high signal output, their applicability has been limited
because of assay matrix-related interference and relatively
high level of nonspecific binding observed in the assays
[1–3]. Matrix effects in an immunoassay usually refer to
interference arising from the sample matrix (e.g., whole blood,
serum, or saliva) that contains the analyte. Previously, we have
used a systematic approach to characterize different aspects of
a sandwich-type non-competitive immunoassay utilizing
nanoparticle bioconjugates as labels . We have developed
a rational basis for development of immunoassays using
nanoparticles as labels [5–7] and now expand this work to describe
assay matrix-derived interactions .
Matrix-related interferences like high nonspecific binding
and cross-reactivity of antibodies, autoantibodies (HAB),
human anti-mouse antibodies (HAMA), and polyanions
(heparin) often hamper precise measurements in clinical
samples, e.g., in serum [9, 10]. In most assays, serum samples are
preprocessed from whole blood by removing the blood cells
and clotting factors, but they still contain a variety of factors
that prevent reaching the lowest limit of detection (LLD) that
can be obtained with artificial buffer samples. Binding of a
nanoparticle bioconjugate is defined by nanoparticle material
and the bioconjugate layer, but also by their biomolecule
corona . The biomolecule corona of a nanoparticle describes
a loosely bound dynamic layer of non-covalently associated
proteins and other biomolecules that in part mediate
nanoparticle interactions . TSH and thyroid hormone assays are
especially prone to HAB interference, as both Graves’ and
Hashimoto’s diseases are autoimmune disorders, implying
the prevalence of autoantibodies to the hormones or their
respective receptors. Thus, optimization of TSH assay
conditions is of particular interest. There are a number of sample
preprocessing strategies for various immunoassays, but none
that would directly focus on removing nanoparticle
We used TSH as a model analyte to demonstrate advances
in immunoassay technology. TSH is a 28-kDa glycoprotein
hormone secreted by the pituitary gland. TSH comprises two
chemically different subunits, α and β, which are joined by
non-covalent bonds. The normal range of TSH in serum is
0.3–5.0 mU L−1 (0.05–0.8 μg L−1), but over 95% of screened
normal euthyroid volunteers have TSH levels below
2.5 mU L−1 . Abnormal TSH level can be a sign of thyroid
malfunction, and it can be used as a tool to diagnose thyroid
diseases and to monitor the effectiveness of therapy. Patients
suffering from hypothyroidism have elevated TSH level, and
more precise TSH assays allow one to distinguish subclasses
of hyperthyroidisms. Over 13 million Americans are believed
to be affected by some type of thyroid disease, but thyroid
diseases nevertheless remain underdiagnosed because of
ineffective screening programs . Furthermore, precise analysis
of thyroid function is required during pregnancy, as one to
three in 200 pregnancies are affected by thyroid dysfunction
caused by the autoimmune disorders Graves ’ and
Hashimoto’s diseases [15, 16]. These conditions are
dangerous and may cause congestive heart failure for the mother,
miscarriage, and attention deficit hyperactivity disorder
symptoms or impaired cognitive development for the child. The
likelihood of adverse effects is increased especially if the
thyroid disease develops during the first trimester [16, 17].
Furthermore, there is increasing evidence on thyroid
dysfunction being one of the factors triggering or aggravating
metabolic syndrome [18, 19]. Slightly elevated TSH levels have
been linked to metabolic syndrome, also in euthyroidism; in
particular, young females with a TSH in the upper normal
range (2.5–4.5 mU L−1) were more likely to be obese, had
higher triglyceride levels, and were more likely to be affected
by metabolic syndrome.
Good precision and accuracy are often problematic to
achieve at the same time, and therefore small changes in trends
cannot usually be detected in a number of diagnostic test
formats. Hence, more powerful tools are required to
quantitatively detect low concentrations of target analyte, small changes in
concentrations, and trend reversals due to intervention therapy
. In plasma and serum the matrix-related interferences
often hamper precise measurements, especially in the case of
autoimmune disorders . In this study we demonstrate the
effects of our conceptual findings on the TSH immunoassay
performance, and especially their potential to reduce matrix
interference. Our optimized europium(III) nanoparticle
labeling technology in sandwich-type immunoassays is shown to
improve the LLD to 60 nU L−1, which equals 450 amol L−1
, and is 1/50 of the LLD obtainable by the current market
leaders in TSH assays. The assay is based on 96 well-plate
format, where Eu(III)-labeled nanoparticles are used as labels
and time-resolved fluorescence is used to monitor TSH
quantitatively from the surface of a reaction well.
carboxylmodified, monodisperse, polystyrene nanoparticles with a
92-nm diameter were acquired from Seradyn (Indianapolis,
IN). The fluorescent properties of these particles were
described previously [22, 23]. The particles are stabile in
aqueous suspensions and nanoparticle material protects doped
Eu-chelates, thereby facilitating stabile fluorescence .
Anti-TSH monoclonal antibodies (clones anti-TSH 5404
SP-1 and anti-TSH 5409 SPTNE-5) were purchased from
Medix Biochemica (Kauniainen, Finland). Antibody
fragments (anti-prostate specific antigen (PSA) Fab 5A10, Fab
anti-TSH 5409, and anti-TSH 5404) were produced at the
Department of Biotechnology in the University of Turku
[25, 26]. Affinity constants for the antibodies were 9.3 ×
108 L mol−1 and 2.2 × 1010 L mol−1 for anti-TSH 5409 and
anti-TSH 5404, respectively . KaivogenSA96™
streptavidin-coated microtitration low-fluor plates, KVG
buffer [50 mmol L−1 Tris–HCl (pH 7.8), 150 mmol L−1
NaCl, 7.7 mmol L−1 NaN3, 76 μmol L−1 bovine serum
albumin, 80 μmol L−1 Tween 40, 3 μmol L−1 bovine γ-globulin,
20 μmol L−1 diethylenetriaminopentaacetic acid] and
washing solution (5 mmol L−1 Tris–HCl, pH 7.8 containing
150 mmol L−1 NaCl, 3.5 mmol L−1 Germall II, and
40 μmol L−1 Tween 20) were from Kaivogen Oy (Turku,
Finland). N-Hydroxysulfosuccinimide (NHS) was acquired
from Fluka (Buchs, Switzerland); bovine serum albumin
fraction V (BSA), biotin, and
N-(3-dimethylaminopropyl)N′-ethylcarbodiimide (EDC) were purchased from Sigma
Conjugations and coatings
Nanoparticles were covalently coated with Mab anti-TSH
5404 according to a previously described procedure using
6 μmol L−1 Mab  (Fig. 1). The monoclonal antibodies
were biotinylated randomly through lysines according to a
protocol described earlier . The Fab fragments were
produced, site-specifically biotinylated, and coated on
streptavidin-functionalized solid phase according to a
previously described procedure . The nanoparticle bioconjugates
were diluted 5- to 1000-fold into KVG buffer or a subset of the
buffer’s components and incubated for at least 24 h to allow
their protein corona to re-equilibrate .
The spot-wells were produced by washing
streptavidincoated microtitration wells twice with the washing solution
to remove preservatives and loosely bound proteins. To
produce fully coated wells 3 × 10−13 mol of biotinylated Fab
fragments were incubated in the prewashed wells in 30 μl of KVG
buffer for 20 min. In spot-coating 1 × 10−13 mol of
biotinylated Fab fragment was incubated in 1 μL of KVG buffer for
10 min. The spot was produced by pipetting a drop of capture
antibody solution halfway between the edge and center of the
well of a streptavidin-functionalized 96-well plate. The
majority of the solid phase is thus left devoid of the capture
antibody, and binding of the nanoparticle bioconjugate on it will
thus be significantly lower (Fig. 1b) .
Known concentration of TSH was spiked in KVG buffer and
incubated for 20 min. Alternatively, the TSH was mixed into
affinity purified serum/KVG buffer mixture and incubated for
10–20 min (Fig. 1). In the one-wash assay configuration TSH
was spiked into a 1:1 mixture of affinity-purified pooled
serum and KVG buffer, and 20 μL of this mixture was then
transferred to a reaction well. After 10 min of incubation under
fast circular shaking (DELFIA Plateshake 1296-003, with a
circular vibrating motion, PerkinElmer), 3 × 107 detector
nanoparticle bioconjugates in 30 μL of KVG buffer were
added to the reaction wells and incubated for another 30 min
under fast circular shaking. Subsequently, the wells were
washed six times and aspirated. The time-resolved
fluorescence from the nanoparticle–antibody bioconjugates was
measured by excitation at 340 nm and detection at 615 nm
using time-resolved fluorescence of plate reader Victor2 1420
Multilabel counter (Wallac, PerkinElmer) .
Serum was isolated from peripheral blood of healthy
volunteers (N = 7; 3 male, 4 female) who gave their informed
written consent. Affinity purification was performed by
incubating pooled human serum in microtiter wells coated with 50 μL
of 100 nmol L−1 anti-TSH Mab 5404 (Fig. 1b). Alternatively,
the serum was purified with an antibody against prostate
specific antigen (PSA) anti-PSA Fab 5A10 (50 μL of
200 nmol L−1) that was, apart from the paratope, structurally
similar to the anti-TSH antibodies (Fig. 1c). In this experiment
a female donor’s serum was used because of lack of PSA in
the circulation. The donors’ (N = 2) TSH was tested to be
below 1 mU L−1 2–4 weeks before the serum sample for this
study was taken. The incubation lasted 15 min, and the serum
was treated twice in a volume of 60 μL. The purified serum
was extracted from wells and stored refrigerated or frozen
until used. We used regular two-way ANOVA with Sidak’s
test for multiple comparisons available in GaphPad Prism to
test significance of the observed difference between
affinitypurified samples and untreated samples.
Dynamic light scattering measurements
The nanoparticle bioconjugates were vortexed, diluted into
KVG buffer, sonicated, vortexed again, after which the
dynamic light scattering (DLS) was measured immediately
(Zetasizer Nano, Malvern Instruments, Worcestershire, UK).
The stabilized samples were diluted, sonicated, vortexed, and
allowed to re-equilibrate their protein corona for 24 h, after
which the samples were sonicated and vortexed again and
DLS was measured.
The colloidal stability of nanoparticle bioconjugates in
suspension is determined by their surface potential , stability
of conjugated antibodies , ionic strength and pH of the
assay buffer , concentration of nanoparticle bioconjugates
in the suspension, detergents in the assay buffer, presence of
blocking proteins, and stability of their protein corona . In
contrast to small molecular labels attached to antibodies that
have a miniscule influence on their microenvironment, the
Fig. 1 Schema and flow diagram of the assay concept. a Sandwich
complex. Carboxyl groups on Eu-doped polystyrene nanoparticles are
functionalized with EDC-NHS (treatment to generate succinimidyl
groups). Then the particles are coated with monoclonal anti-TSH
antibodies to form the nanoparticle bioconjugates and unreacted succinimidyl
groups are converted back to carboxylic acid through hydrolysis. The
solid phase is first coated with streptavidin and blocked with BSA;
subsequently, an area of approximately 1 mm2 is functionalized with
antiTSH Fab fragments recognizing a different epitope in TSH than the
nanoparticle-conjugated antibodies. b To optimize the assay we needed
TSH-free human serum and we produced it by affinity purification of
pooled human serum. For the purification we used 2 × 10 min incubation
of the serum in anti-TSH-coated microtiter wells, after which we spiked
samples with known concentrations of recombinant human TSH. The
TSH was added to serum into equal volume of KVG buffer, and 20 μL
of this mixture was added to spot-coated wells. After 10 min incubation
with fast circular mixing the nanoparticle bioconjugates were added in a
volume of 30 μL and incubation was continued for 30 min. Subsequently,
unbound nanoparticle bioconjugates were washed away, and wells were
read for time-resolved Eu-fluorescence. c To reduce nonspecific binding
in a nanoparticle-based immunoassay we used a nonspecific affinity
purification step to remove compounds interfering with the assay. First, a
sample of female serum low–normal range TSH sample was spiked with
100 μU L−1 of TSH in KVG buffer (Fig. 4) and incubated for 2 × 10 min
in anti-PSA Fab-coated wells (women do not have PSA in circulation so
nothing specific is removed). A replicate sample was spiked, but left
untreated. 20 μL of both samples were transferred to spot-coated wells
and assayed like in protocol b. Difference in TSH-assay performance is
presented in Fig. 4
nanoparticles form a polarized solvation shell near the surface
of the particle where exchange of molecules will be slower
. As the nanoparticle bioconjugates are typically diluted in
the assay buffer from a separate storage buffer, i.e., stock
suspension, the equilibrium of proteins loosely adhered to
their surface is perturbed. During formation of the new
equilibrium and molecule exchange on the nanoparticle
surface, low energy bonds (e.g., electrostatic and van der
Waals) form and break, and this may generate unwanted
nonspecific interactions [31–33]. We discovered that
storing nanoparticle bioconjugates in KVG buffer (a buffer with
blocking proteins and detergent) at a concentration of no
higher than 100 times that of the final usage concentration
in the following assay increased the signal-to-noise ratio of
the assay by nearly threefold (Fig. 2a). This can be explained
by agglomeration of nanoparticle bioconjugates upon
coming into contact with the high protein concentration in the
serum matrix  or the assay buffer, as they are diluted
from a high storage concentration. We examined the process
by dynamic light scattering (DLS) experiments where
nanoparticle bioconjugates were diluted in KVG buffer and
measured immediately or after 24 h protein corona stabilization.
We observed an increase of hydrodynamic radius of single
particles corresponding to the adsorption of matrix proteins
onto the nanoparticle bioconjugates and formation of a new
protein corona (Fig. 2b). Furthermore, we observed both
fewer and smaller aggregates in the stabilized samples
(Fig. 2c). The aggregates represented a particle volume
below 5% of the total DLS signal; hence, these aggregates
were difficult to measure, resulting in high variation of
signal between the replicate samples. The controlled
reformation of the protein corona (Fig. 2b) appears to reduce
agglomeration-inducing interactions between particles
(Fig. 2c), and as a result the immunoassay had higher
specific signal, as more binders were available, and lower
nonspecific signal (Fig. 2a), as prevalence of large aggregates
prone to sediment is reduced.
Components in the protein corona may improve colloidal
stability and increase the activity of the nanoparticle
bioconjugates by reducing unwanted interactions or decrease their
Fig. 2 Concentration-dependent protein corona stabilization. a The
nanoparticle bioconjugates were stored in KVG buffer at various
concentrations and then used to perform a one-step assay for 100 μU
L−1 TSH in KVG buffer assay matrix. Dilution to the assay concentration
(2.5 × 106 particles μL−1) was made in a single step. Stabilization of the
particle suspension was achieved up to a concentration 100-fold higher
than the concentration used in the assay. Mean values of three replicates
are presented as bars and standard deviation as error bars. b Main peak of
a DLS measurement of nanoparticle bioconjugates diluted into KVG
buffer measured immediately after dilution, sonication, and vortexing
(black circles, black line represents a mean of 3 replicates) and one
measured after dilution, 24 h stabilization, sonication, and vortexing (red
squares, deep red line represents a mean of 3 replicates). c
Representation of the aggregate peak of the same DLS measurement;
while we observed aggregates only in some of the samples, typically
the aggregates were fewer and smaller in the stabilized samples. A zero
value indicates that the amount of aggregates in the sample fell below the
binding activity by masking binding sites . To investigate
the stabilization effect in more detail we did a
component-bycomponent test of the KVG buffer ingredients to discover the
components responsible for the effect. Also, we wanted to
reveal whether the effect was due to more dispersed particle
suspension after the dilution rather than due to any specific buffer
components. The test of the buffer ingredients was performed at
a particle concentration of 1.25 × 108 particles μL−1 (Fig. 3) 
. The most critical storage buffer component regarding the
stabilization was the blocking protein BSA, and the second most
important was detergent Tween 40. The importance of the
detergent was less pronounced when buffer pH and salinity, i.e.,
ionic strength, were adjusted to physiological range.
Stabilization using the optimum pH, physiological ionic
strength, blocking protein, and detergent in the diluted particle
suspension resulted into a nearly threefold improvement in
signal-to-background ratio. However, when these components
were added to the storage buffer at a particle concentration
1.25 × 1010 particles μL−1, no benefit was observed. This
would indicate that the stabilization effect is dependent on the
surface kinetics, as increasing the concentration of stabilizing
agents to match the surface area did not provide a similar result.
Control of association time by solid-phase organization
We have shown with force spectroscopy and surface
plasmon resonance that the association rate of specific binding
of nanoparticle bioconjugates (kon) is up to 200,000-fold
higher than kon for nonspecific binding, while koff for both
Component-bycomponent investigation of the
storage buffer composition at
concentration of 1.25 × 108
particles μL−1 and 1 mU L−1 of
TSH. Mean values of three
replicates are presented as bars
and standard deviation as error
bars. Impact of the storage buffer
composition is measured by
testing the assay performance
(signal-to-background ratio) with
the particles stored in the
specified buffer and then diluted
into KVG buffer. The best
signalto-background ratio was obtained
with particles stored in
50 mmol L−1 Tris–HCl (pH 7.8),
150 mmol L−1 NaCl, 76 μmol L−1
bovine serum albumin, and
80 μmol L−1 Tween 40
specific and nonspecific binding was too slow to be
measured . Also, nonspecific binding occurs mainly between
two antibody-coated surfaces  and is dependent on the
density and stability of the antibodies . We were able to
show that colloidal stability is critical for assay performance
(Fig. 2); to further reduce sedimentation and nonspecific
binding, we decided to reduce the binding time of the
nanoparticle bioconjugates with fast liquid flow induced by
mixing and a small area for specific binding (Figs. 1 and
4) [35, 36]. The small area of the active solid phase was
produced by adding the Fab capture antibody in a 1-μL drop
on a well of a streptavidin-functionalized 96-well plate,
halfway between the edge and center of the well. At this
location the liquid flow induced by circular shaking was
the fastest and no edge effect could be expected. Thus, the
majority of the solid phase was left devoid of the capture
antibody. Binding of the nanoparticle bioconjugate towards
the streptavidin-coated and BSA-blocked surface was 10–
500 times less efficient than to antibody-coated surface [5,
23]; hence, a lower level of background signal was observed
with zero TSH calibrator samples. The spot-coated wells
provided 3- to 10-fold higher signal-to-background ratio
than the fully coated wells (Fig. 4). Moreover, the assays
in spot-coated wells required lower concentration
nanoparticles to produce optimal signal level (see Electronic
Supplementary Material (ESM) Fig. S1), because we
directed the excitation beam of the Victor2-multilabel counter to
the coated spot and achieved a strong excitation of the
specifically bound nanoparticle bioconjugates. The nonspecific
Fig. 4 Comparison of TSH assay signal-to-background ratios in KVG
buffer (buffer, gray bars) and in 50% affinity-purified serum (serum,
black bars). TSH concentration was 100 μU L−1; mean values of three
replicates are presented as bars and standard deviation as error bars. The
assay utilized normal (ctrl-NP) and protein corona-stabilized nanoparticle
bioconjugates (s-NP) and fully coated (FC) and spot-coated (spot)
microtiter wells. The optimized configuration gave 10-fold higher
signal-tobackground ratio in KVG buffer configuration with fully coated well
and control NPs and a 100-fold higher ratio in 50% affinity-purified
serum configuration with fully coated well and control NPs
signal is directly proportional to the number of nanoparticle
bioconjugates used in the assay, so this in part helped to
reduce the background signal .
Sample preprocessing by affinity purification
The normal range of TSH in patient samples is approximately
0.4–2.5 mU L−1. To measure a TSH calibration curve, the
serum samples were prepared from pooled serum samples
by affinity purification with anti-TSH antibodies, and
subsequently these samples were spiked with recombinant TSH
(Fig. 1b). We observed that the Eu-signal measured from zero
TSH calibrator of TSH-stripped serum samples was lower
than that measured from buffer-only samples (Fig. 4).
Analyzing these samples we observed a reduced and
reproducible level of nonspecific background signal in the zero
TSH calibrator sample (Fig. 4). This implies that the protocol
used to remove TSH from the pooled serum also removed
other cross-reactive or interfering compounds from the
sample. Removal of such compounds from a clinical sample
would naturally enable detection of even lower concentrations
of TSH. We then asked if it would be possible to remove these
interfering compounds with a pretreatment using an antibody
that does not recognize anything specific in the serum
samples. To quantify this observation, we utilized anti-PSA 5A10
Fab fragment -coated wells to purify pooled female serum
samples with low-normal range TSH (0.4–1 mU L−1) after
they had been spiked with (100 μU L−1) recombinant TSH
(Fig. 1c). The anti-PSA Fab was produced in a similar manner
as the anti-TSH Fab fragment, and the amino acid sequence
was over 90% similar. However, in female serum samples
there should not exist any epitopes that the anti-PSA Fab
fragment would recognize, and therefore the observed affinity
purification was not caused by removal of any specific
interacting compound. After the purification step, we
measured recovery of spiked recombinant TSH 100 μU L−1 in
samples with 50%, 20%, 10%, and 5% of serum (Fig. 5).
The TSH concentration of all the samples was equal, and only
the amount of serum differed. Therefore, with perfect recovery
of recombinant TSH, the signal in all measurements should be
equal to 5% sample. The observed decrease of Eu-signal
represents reduced TSH recovery due to matrix-related effects.
We observed that at serum quantity over 5% (v/v) the affinity
purification produced a significant increase of TSH recovery
(Fig. 5). The effect of affinity purification was stronger the
higher the proportional amount of serum in the sample was.
We used our findings to develop a heterogeneous
sandwichtype immunoassay utilizing nanoparticle bioconjugates to
measure TSH in affinity-purified pooled human serum. A
standard curve measured using three replicates of each
calibrator reached an LLD of 60 nU L−1 (Fig. 6); this corresponds
to 450 amol L−1 or 10,000 molecules in a sample volume of
Fig. 5 Affinity purification increases the sensitivity of the assay. We
added (100 μU L−1) of TSH (Sample + 100 μU L−1) into female serum
samples with low–normal range TSH (Sample) (approximately 0.4–
1 mU L−1). TSH recovery was illustrated by plotting Eu-signal
(Sample + 100 μU L−1) – Eu-signal (Sample). The serum samples were
not purified (black) or affinity purified (gray) with Fab 5A10 anti-PSA
antibodies that beared a resemblance to anti-TSH antibodies, but having
no specific antigens in the serum. Addition was done in respect to total
well volume, and thus all samples should have given the same Eu-signal.
Differences between treated and untreated samples were tested with
regular two-way ANOVA with Sidak’s test for multiple comparisons; ns p >
0.05, **p < 0.01, and ***p < 0.001
Fig. 6 TSH standard curve measured in spiked affinity-purified serum
samples by using three replicates of each calibrator. The LLD of the assay
was 60 nU L−1 corresponding to 450 aM or 10,000 molecules in sample
volume of 20 μL. Background signal was subtracted from the data points
presented and the average background signal is set to zero value and LLD
at 3 × SD of background
20 μL. The LLD was defined as the signal of 3 × SD of the
zero calibrator over the signal obtained from zero calibrator.
Furthermore, in the presented assay the slope of signal
increase for each 1 μU of TSH in serum was 13,500 Eu-signal
units. The high sensitivity (signal increase per unit of TSH) of
the assay enabled high precision in the determination of a
patient’s TSH level.
The assay is a one-wash configuration where the undiluted
sample is first dispensed into a microtiter well and thereafter
tracer nanoparticles are added to the same well without a
separation step in between. A single washing step is required
before the time-resolved luminescence signal is recorded.
The presented assay is relatively easy to perform, total time
to conduct the assay is 40 min, and it requires low amounts of
sample and reagents, although the affinity purification
increases slightly with the amount of antibodies used. We were
able to operate the assay with serum volumes of 5–40 μL.
Further reduction of sample volume induced a non-linear
reduction in measured LLD. There was no kinetic requirement
for the incubation period, but we chose to measure the signal
when equilibrium was reached for the binding reaction
because we wanted the principles presented to be more
Our longstanding aim has been to establish general
principles for developing sandwich-type immunoassays utilizing
Eu-nanoparticle labels [4–8, 23]. Here we applied those
principles, focusing on the matrix-related nonspecific
binding and developed a sandwich-type heterogenic
immunoassay for TSH that has a lower LLD and higher sensitivity
than previously presented assays. The LLD of 60 nU L−1
in our assay is nearly 1/50 of that of the market-leading
assay systems: the chemiluminescence microparticle
immunoassay (CMIA) 2.5 μU L−1 by Architect i2000 SR
(Abbott Diagnostics) or the electrochemiluminescence
immunoassay (eCLIA) 5 μU L−1 by Cobas 6000 (Roche
Diagnostics). Furthermore, we introduce a concept of
stabilization of the protein corona surrounding the
nanoparticle bioconjugates that reduced nonspecific binding,
especially in clinically relevant matrixes. The presented assay
concept will facilitate better recovery of TSH from serum
and more precise TSH measurements, and thereby smaller
sample volumes. The observed increase in recovery
suggests that there are components preventing interaction of
n a n o p a r t i c l e l a b e l a n d T S H i n t h e s e r u m m a t r i x .
Furthermore, the observed decrease of background signal
after the antibody capture-based removal of TSH implies
that some of these interacting components cause the
increase of nonspecific binding. Although we have not
defined the interacting compounds, we suggest that a
preprocessing step comprising an affinity purification with an
antibody not binding the analyte or matrix filtration (Fig. 5)
would increase the performance of many clinical
immunoassays and would help to remove both false positives and
negatives [20, 37, 38]. We hypothesize that removal of
interfering compounds would be especially important in
samples where HABs are likely to be present . Furthermore,
we propose that the best result of affinity purification is
likely to be produced by an antibody resembling the capture
antibody that has structural differences only in the
hypervariable loops of the paratope.
The assay performance was further improved with of
spotcoated configuration of the active solid phase and stabilized
protein corona surrounding the nanoparticle bioconjugates.
Dilution of the nanoparticle bioconjugates into another buffer
or assay matrix causes a change in their protein corona, i.e.,
proteins loosely adhering to the nanoparticles [12, 33]. Rapid
change in the protein corona may cause aggregation of the
nanoparticles, as macromolecules in the matrix transiently
interact with multiple nanoparticles. These interactions may also
mask some of the binding sites, if a dense corona is
electrostatically attracted to surround a nanoparticle (Figs. 2 and 3)
. These effects can be avoided by incremental dilution of
nanoparticles and maintaining adequate concentration of
stabilizing compounds (Fig. 3) and allowing time for the protein
corona to stabilize before the assay in conducted (Fig. 2).
Moreover, we provide evidence that the stabilization effect is
independent on nanoparticle concentration and that
reformation of the protein corona can be observed with DLS
(Fig. 2). The benefit of spot-coated solid-phase configuration
(Figs. 4 and 6) is likely to originate from reduced rolling of
nanoparticles on an antibody-coated surface  that would
potentially increase nonspecific binding by allowing more
time for the nanoparticle bioconjugates to adhere via
nonspecific bonds. Also, the configuration concentrates the
analyte in a confined area promoting efficient multivalent
recognition by the nanoparticle. Yet another mechanism through
which an improved signal-to-background ratio could be
mediated is the decrease in the optimal amount of nanoparticle
bioconjugates needed for the assays done in spot-coated wells
(see ESM Fig. S1). Our assay concept, however, could not be
applied to heparin plasma, most likely because of interactions
caused by the polyanions. The ability to perform a
washingfree immunoassay in a whole blood sample smaller than
20 μL can be seen as a prerequisite for patient self-testing
and point-of-care systems testing for biomarkers in the
circulation . While we present a heterogeneous test, the LLD
measured is sufficient to design a separation-free or
homogenous system that typically performs at an order of magnitude
higher LLD or better. We suggest that the principles found in
this study are applicable to, e.g., lateral flow-based systems.
The higher the sensitivity of the assay is, the smaller the
concentration differences that can be measured. Such a
property is of importance when assessing drug response and
appropriate dosage, especially in rapidly developing conditions
like pregnancy-related thyroid dysfunctions where both too
high and too low thyroid hormone concentrations pose a risk
for the fetus and the mother [16, 17, 40]. These conditions
require exact and repeated measurements to adjust the drug
dosage over the course of pregnancy , and in some cases
treatment needs to be started to promote fertility .
Furthermore, thyroid hormones modulate many metabolic
pathways relevant to the resting energy expenditure, and
hypothyroidism is associated with weight gain and metabolic
syndrome . Recognizing trend reversals is essential in
the treatment of metabolic syndrome [19, 43].
A super-sensitive time-resolved fluoroimmunoassay for TSH,
LLD 60 nU L−1, utilizing europium(III) nanoparticle labels
with reduced nonspecific binding was developed. An affinity
purification step with an antibody bearing close resemblance
to the detecting and capture antibody removed interfering
compounds from the sample matrix . Combining these
properties with previously found general parameters in
sandwich immunoassays [5–7] facilitated detection of TSH
concentrations that were 1/50 of LLD of the current
marketleading technologies (Fig. 6) and increased sensitivity .
With more accurate diagnostics, intervention therapy can start
earlier, and total health care costs can be reduced. However,
precision and accuracy are often problematic in a number of
test formats, and small changes in trends cannot be detected.
There is continuing demand for high-performance clinical
tests, and central laboratories still process the majority of
diagnostic tests owing to lower unit costs [39, 44].
Acknowledgements The authors gratefully acknowledge the Academy
of Finland for funding the research under grants #110174, #260599 and
The Finnish National Doctoral Programme in Informational and
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Compliance with ethical standards Serum was obtained from healthy
volunteers who gave their informed written consent. The study was
approved by the Ethics Committee of Southwest Finland Hospital District,
and was carried out in accordance with the guidelines set for research on
human samples by Turku University Hospital and University of Turku.
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