Document [original]

Vol.:(0123456789)

1 3

https://doi.org/10.1007/s00216-021-03778-7

RESEARCH PAPER

A rapid magnetic bead‑based immunoassay forsensitive

determination ofdiclofenac

AlexanderEcke1,2· TanjaWestphalen1· JaneHornung3· MichaelVoetz3· RudolfJ.Schneider1,4

Received: 25 August 2021 / Revised: 18 October 2021 / Accepted: 5 November 2021

Abstract

Increasing contamination of environmental waters with pharmaceuticals represents an emerging threat for the drinking

water quality and safety. In this regard, fast and reliable analytical methods are required to allow quick countermeasures

in case of contamination. Here, we report the development of a magnetic bead-based immunoassay (MBBA) for the fast

and cost-effective determination of the analgesic diclofenac (DCF) in water samples, based on diclofenac-coupled mag-

netic beads and a robust monoclonal anti-DCF antibody. A novel synthetic strategy for preparation of the beads resulted

in an assay that enabled for the determination of diclofenac with a significantly lower limit of detection (400ng/L) than

the respective enzyme-linked immunosorbent assay (ELISA). With shorter incubation times and only one manual wash-

ing step required, the assay demands for remarkably shorter time to result (< 45min) and less equipment than ELISA.

Evaluation of assay precision and accuracy with a series of spiked water samples yielded results with low to moderate

intra- and inter-assay variations and in good agreement with LC–MS/MS reference analysis. The assay principle can be

transferred to other, e.g., microfluidic, formats, as well as applied to other analytes and may replace ELISA as the standard

immunochemical method.

Keywords Immunoassay· Magnetic beads· Diclofenac· Water analysis· LC–MS/MS

Introduction

The nonsteroidal anti-inflammatory drug (NSAID)

diclofenac (DCF, Fig.1) has been used frequently in

the treatment of rheumatic diseases, inflammations,

and fever, as well as acute and chronic pain since its

introduction in the 1970s [1, 2]. Together with ibupro-

fen and naproxen, DCF is among the most commonly

sold non-aspirin NSAIDs in several countries worldwide

over the past years [3, 4]. Accordingly, the discharge

of DCF via wastewater is relatively high considering

that the majority of orally administered DCF is excreted

unaltered or metabolized via urine (65–70%) or feces

(20–30%) and DCF applied cutaneously is mainly washed

away (> 90%) [5, 6].

Insufficient degradation in wastewater treatment plants

(WWTPs) promotes introduction of DCF into surface waters

where its ecotoxicological effects can lead to disruption of

whole biosystems [6–12]. As a consequence, DCF has been

proposed as a priority substance and was added to the EU

watchlist for substances of concern requiring Union-wide

monitoring in the field of water policy [13]. In this context,

environmental quality standards for DCF in inland waters

of 100ng/L and 10ng/L in all other surface waters were

proposed [14]. The concentration values of DCF found in

various surface waters, however, exceed these limits with

values in the low µg/L range reported for rivers in Europe,

Asia, Africa, America, and even Antarctica (for a review, see

[12]). As a result, DCF can also reach water bodies that are

used for drinking water preparation which is highly alarm-

ing in concern of human health. So far, concentrations in the

two- to three-figure ng/L-range are already found in many

groundwater and drinking water samples [12]. Strategies for

* Rudolf J. Schneider

r[email protected]

1 Department ofAnalytical Chemistry; Reference Materials,

Bundesanstalt für Materialforschung und -prüfung (BAM),

12489Berlin, Germany

2 Department ofChemistry, Humboldt-Universität zu Berlin,

12489Berlin, Germany

3 sifin diagnostics gmbh, 13088Berlin, Germany

4 Technische Universität Berlin, Faculty III Process Sciences,

10623Berlin, Germany

/ Published online: 20 November 2021

Analytical and Bioanalytical Chemistry (2022) 414:1563–1573

1 3

more effective degradation of DCF in WWTPs to prevent

introduction into environmental water, and thus into drink-

ing water, have been reported before [15–20], but are not yet

applied comprehensively so that fast and reliable analysis

methods combined with local countermeasures in case of

contamination are still required.

The method of choice for the determination of DCF in

water samples with high accuracy is LC–MS/MS in com-

bination with solid-phase extraction for sample preparation

[13, 21]. However, these methods are time-consuming and

expensive, and require specially trained personnel. In this

regard, immunoassays based on the binding of the respec-

tive analyte to an analyte-selective antibody and the quanti-

fication of these binding events have gained importance as

they enable fast (high-throughput) and cost-effective (low

amounts of reagents required) analyses. Moreover, thanks

to portable detection units, measurements can also be per-

formed outside of the lab at the point of care.

First and foremost, a specific antibody is needed for

effective immunochemical detection. Several anti-DCF anti-

bodies with different affinities and sensitivities have been

developed in recent decades [22–24]. Based on this, immu-

noassays were set up in the well-investigated and validated

ELISA format to investigate binding conditions and cross-

reactivity [22, 23, 25]. For on-site application, though, the

conventional microplate-based ELISA is still too lengthy

and dependent on bulky equipment like shakers, washers,

and microplate readers. This is why efforts have been made

to transfer the immunochemical detection to faster and/or

more mobile platforms. Faster immunoassays developed

for DCF comprise an upconversion-linked immunosorb-

ent assay (ULISA) [26], a fluorescence polarization immu-

noassay (FPIA) [27], and a suspension array fluorescence

immunoassay (SAFIA) [28]. Steps forward to mobile and

automated sensing of diclofenac have been made with sev-

eral immunosensors for the detection of DCF reported in

recent years [29–35].

In this context, the application of magnetic parti-

cles appears promising as these can be immobilized and

released easily by applying an external magnetic field.

By coupling one component of the immunoassay to the

particle surface, the assay could be implemented into an

immunosensor for automated online detection as shown for

other analytes previously [36–38]. Usually, the antibody

is immobilized on the bead surface (direct approach) to

set up a magnetic bead-based assay which is often more

straightforward [39–42]. For some analytes, however, cou-

pling of the analyte molecule to the particle surface (indi-

rect approach) is more feasible [43–45]. For DCF, it could

be mandatory. To date, no direct immunoassay for DCF

has been reported. Concerning peroxidase-based immu-

noassays, the reason for this may lie in DCF’s property of

serving as a substrate for peroxidase [15]. Because a direct

immunoassay for DCF would require an enzyme tracer, i.e.,

a conjugate of DCF coupled to peroxidase, this may be the

main obstacle as the effects of coupling an enzyme to its

substrate are not yet studied in detail.

This is why we chose an indirect approach to set up an

MBBA for the quantification of DCF in water samples with

the robust anti-DCF antibody F01G21 [24]. A novel syn-

thetic strategy for DCF coupling to the bead surface was

used to tune binding properties of the antibody in order to

allow for a more sensitive detection. In combination with the

advantages of magnetic particles, a fast and reliable immu-

noassay with potential for further application, e.g., imple-

mentation into an immunosensor, is presented.

Materials andmethods

General equipment

Ultrapure water was taken from a Merck Millipore (Darm-

stadt, Germany) Milli-Q Reference water purification sys-

tem. Weighing was performed on a Sartorius (Göttingen,

Germany) Research R180D-*D1 or Cubis® Advanced

MCA225S-2S00-I analytical balance. Adjustment of pH

values was done with a SevenEasy pH meter S20 from Met-

tler Toledo (Columbus, OH, USA).

Preparation ofDCF‑coupled beads

Bead preparation and all related reactions were performed

in 2-mL centrifuge tubes from Eppendorf (Hamburg, Ger-

many). Shaking of reaction mixtures was executed in a Ther-

moMixerC from Eppendorf, and centrifugation was carried

out using an Eppendorf Centrifuge 5417R. For washing, a

BioMag® Multi-6 Microcentrifuge Tube Separator from

Polysciences (Hirschberg an der Bergstraße, Germany) was

used.

NHS‑activation ofDCF

As reported before [28], diclofenac sodium salt (Sigma-

Aldrich, Steinheim, Germany) was dissolved in dry N,N-

dimethylformamide (DMF, Sigma-Aldrich) under argon

atmosphere to a final concentration of 1/6mol/L. Stock

Fig. 1 Chemical structure of

diclofenac (DCF)

1564 Ecke A. et al.

1 3

solutions of N-hydroxysuccinimide (NHS, Merck) and

N,N’-dicyclohexylcarbodiimide (DCC, Sigma-Aldrich) of

1/2mol/L each in DMF were prepared under argon. To the

DCF solution, NHS solution (1.2eq), a spatula tip of N,N’-

disuccinimidyl carbonate (DSC, Sigma-Aldrich), and DCC

solution (1.2eq) were added in this particular order under

argon. The resulting solution was shaken in the dark at RT

and 750rpm for 18h.

Afterwards, the mixture was centrifuged at 4°C and

4000rpm for 10min in order to separate the precipitated

dicyclohexylurea. The supernatant solution was used directly

for coupling.

Coupling tomagnetic beads

A suspension of amino-functionalized magnetic micropar-

ticles (Sigma-Aldrich, 100µL) in 500µL absolute ethanol

(Th. Geyer, Renningen, Germany) and 500µL 0.1M sodium

bicarbonate (Sigma-Aldrich) was prepared. Consecutively,

100µL of a solution of 0.5mol/L glutaric anhydride (Merck)

in absolute ethanol and 5µL of the above described DCF

active ester solution were added. The resulting mixture was

shaken at RT and 900rpm for 20h.

Thereafter, beads were washed once with Milli-Q water

(1mL) and thrice with absolute ethanol (1mL) using a mag-

netic separator to hold the beads while removing the super-

natant. Beads were then resuspended in absolute ethanol

(1mL) and stored at 4°C until further use.

Buffers

All buffers were prepared in Milli-Q water and stored in

amber glass bottles at room temperature (RT, 22 ± 1°C)

unless stated otherwise. The pH values were adjusted using

6M hydrochloric acid (Merck) or 5M sodium hydroxide

solution (J.T.Baker, Phillipsburg, NJ, USA).

• Phosphate-buffered saline (PBS), pH7.6: 10mM sodium

phosphate monobasic dihydrate (Sigma-Aldrich), 70mM

sodium phosphate dibasic dihydrate (Sigma-Aldrich),

145mM sodium chloride (Sigma-Aldrich).

• Washing buffer, pH7.6: 0.75mM potassium phosphate

monobasic (Sigma-Aldrich), 6.25mM potassium phos-

phate dibasic (Sigma-Aldrich), 0.025mM potassium

sorbate (Sigma-Aldrich), 0.05% Tween20 (Serva, Hei-

delberg, Germany).

• Assay buffer (Tris–EDTA), pH7.6, storage at 4°C:

125mM tris(hydroxymethyl)-aminomethane (Tris,

Merck), 187.5mM sodium chloride, 13.375mM eth-

ylenediaminetetraacetic acid disodium salt dihydrate

(Na2EDTA·2H2O, Sigma-Aldrich).

• Citrate buffer, pH4.0, storage at 4°C: 220mM sodium

citrate monobasic (Sigma-Aldrich).

• TMB stock solution in dry N,N-dimethylacetamide

(DMA, Sigma-Aldrich), storage under argon at 4°C:

8 mM tetrabutylammonium borohydride (Sigma-

Aldrich), 40mM 3,3’,5,5’-tetramethylbenzidine (TMB,

Serva).

Immunoreagents

Polyclonal sheep anti-mouse IgG (H + L chain) antibody

with horseradish peroxidase (HRP) label (secondary anti-

body, R1256HRP) was obtained from OriGene Technologies

(Rockville, MD, USA). Mouse anti-DCF antibodies (isotype

IgG1) F01G21 and SK60-2E4 were produced by fusion of

mouse myeloma cells (AG8 [24] or SP2/0-AG14, respec-

tively) and splenocytes from BALB/c mice, both obtained

from the University of Salzburg [24].

The antibody F01G21 was further labeled with HRP

using the periodate method as described by Wilson and

Nakane [46]. Required chemicals sodium carbonate, sodium

bicarbonate, and ammonium sulfate were from Carl Roth

(Karlsruhe, Germany); sodium periodate and sodium cyan-

oborohydride from Sigma-Aldrich, ethylene glycol from

Serva, and HRP from Roche (Basel, Switzerland). The

antibody solution was concentrated to about 5mg/mL in

an Amicon® Stirred Cell Model 8010 (10mL) equipped

with an Ultrafiltration Disc (30kDa) from Merck Millipore

(Burlington, MA, USA). The labeling reaction was carried

out in a glass vial equipped with a magnetic stirrer. The

labeled antibody was purified in PBS using PD-10 or PD

MiniTrap desalting columns with Sephadex G-25 resins

from GE Healthcare (Chicago, IL, USA). Antibody concen-

tration (2.79mg/mL) and coupling ratio (2.65) were deter-

mined by UV/Vis absorption measurements in UV cuvettes

micro from Brand (Wertheim, Germany) with a BioMate3

UV–Vis Spectrophotometer from Thermo Fisher Scientific

(Waltham, MA, USA). The product was stabilized with

0.04% thiomersal (Serva) and 2% fetal bovine serum (Life

Technologies, Carlsbad, CA, USA), and stored in aliquots

at 4°C for up to 1month or at –20°C for long-term storage.

Standards andsamples

Firstly, a stock solution of DCF-Na analytical standard

(Sigma-Aldrich) in absolute ethanol with a mass concen-

tration of approx. 1g/L was prepared gravimetrically by

weighing both solid and the solvent. From this solution,

serial dilutions in Milli-Q water were made volumetrically

to prepare DCF standards in the concentration range from

10mg/L to 1ng/L.

Spiked water samples were prepared by pre-diluting the

stock solution of DCF-Na analytical standard in Milli-Q

water and successive dilution in the respective sample to the

1565A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac

1 3

desired concentration. Water samples were taken as speci-

fied in Table1.

Immunoassay procedure

All assays were carried out in transparent 96-well flat bot-

tom non-binding polystyrene microplates from Corning

(Corning, NY, USA). Wells were filled using Eppendorf

Research® pro multichannel pipettes. Dilutions were pre-

pared with Eppendorf Research® plus piston stroke pipettes.

Incubation was performed on a Titramax 101 orbital shaker

from Heidolph Instruments (Schwabach, Germany). For

washing, a BioMag® 96-Well Plate Separator from Poly-

sciences was used. Absorbance measurements were carried

out on a SpectraMax® Plus 384 or SpectraMax® i3x micro-

plate reader from Molecular Devices (San José, CA, USA).

For one 96-well plate, 150µL of DCF-coupled beads sus-

pension was mixed with 4.8mL of assay buffer. A volume of

50µL of the resulting suspension was added to each cavity

of the microplate. To this, 100µL/well of DCF standard

solution or sample was added. The HRP-labeled mouse anti-

DCF antibody F01G21 was diluted in assay buffer to a con-

centration of 37.2µg/L, and 50µL of this solution was added

to each well. Then, the resulting mixture was incubated at

RT and 900rpm for 20min.

To remove unbound antibody, washing was performed

by placing the microplate on a magnetic separator. After

waiting for 120s for the particles to separate from the sus-

pension, the supernatant solution was carefully removed by

gentle pipetting and replaced by 200µL of washing buffer.

Subsequently, the plate was shaken for 30s at 1050rpm for

complete resuspension of the particles and the previous steps

were repeated twice.

Still on the magnetic separator, 200µL/well of freshly

prepared substrate solution (22mL citrate buffer, 8.5µL

hydrogen peroxide solution (30%, Sigma-Aldrich) and

550µL TMB stock solution) were added. After the addition

was completed, the plate was shaken for 15min at RT and

900rpm (1050rpm for the first 30s for resuspension of the

beads). Blue color developed in wells with low concentra-

tion of DCF.

Color development was stopped by placing the plate on

the magnetic separator and adding 100µL/well of 1M sul-

furic acid (J.T.Baker) immediately. Color change from blue

to yellow was observed. The plate was further shaken at RT

and 750rpm for 30s, while particles remained separated on

one side of the respective well.

Detection was performed by reading the optical density at

RT at a wavelength of 450nm with reference at 620nm. For

calibration, O.D. values were plotted against the concentra-

tion of standard solutions and a four-parameter logistic func-

tion was fitted to the data points. Concentrations of samples

(24 per plate, each analyzed in triplicate) were determined

by correlating O.D. values to the respective concentration

of DCF standards (8 per plate in triplicate).

LC–MS/MS analysis

LC–MS/MS measurements were carried out on an Agilent

1260 LC system from Agilent Technologies (Waldbronn,

Germany) equipped with a binary pump (G1312B), column

oven (G1316A), autosampler (G1367E), and a diode array

detector (G1315D) coupled to a Triple Quad 6500 Mass

Spectrometer from AB Sciex Instruments (Darmstadt, Ger-

many). A Kinetex® 2.6µm XB-C18 100Å LC column

(150 × 3mm) from Phenomenex (Aschaffenburg, Germany)

and a matching pre-column were used.

Each water sample was analyzed undiluted in duplicate

with an injection volume of 10µL and the column oven tem-

perature set to 55°C. At a flow rate of 350µL/min, a binary

gradient consisting of (A) water and (B) methanol (LC–MS

grade, Biosolve, Valkenswaard, Netherlands) both contain-

ing 10mM ammonium acetate (Sigma-Aldrich) and 0.1%

(v/v) acetic acid (Fluka, Buchs, Switzerland) was used under

the following conditions: 70% A isocratic for 3min; linear

decrease to 5% A within 9min; kept at 5% A for 6min;

increase to 70% A within 0.5min; kept at 70% A for 6min.

Electrospray ionization (ESI) was performed in positive

mode at a source temperature of 400°C and an ionspray

voltage of 4500V. Gas pressures were applied as follows:

curtain gas 35psi, nebulizer gas 62psi, turbo gas 62 psi,

collision gas 8psi. At an entrance potential of 10V, a

Table 1 Details on water samples and sampling procedure

Sample Sampling day Sample description Sampling site

Pure water 08.06.2021 From water purification system BAM building 8.05, room 395C

Drinking water 08.06.2021 From water cooler BAM building 8.05

Mineral water 09.06.2021 Bottled water, nonsparkling, Lichtenauer Pur, BBD: 31.08.21 -

Tap water 08.06.2021 From laboratory water tap BAM building 8.05, room 394

Groundwater 07.06.2021 From water well with an electric pump Groß-Kienitz, 15831 Blankenfelde-Mahlow

Surface water 08.06.2021 From Teltowkanal (canal), filtrated through a 0.45-μm

regenerated cellulose filter Ernst-Ruska-Ufer, 12489 Berlin

1566 Ecke A. et al.

1 3

declustering potential of 90V, a cell exit potential of 15V,

and a dwell time of 100ms for each transition, the mass tran-

sitions m/z 296 → 250 and m/z 296 → 214 with a collision

energy of 22V (m/z 296 → 250), and 30V (m/z 296 → 214)

were used for quantification in selected reaction monitor-

ing mode. For calibration, DCF standard solutions of eight

different concentrations in the range from 0.2 to 100µg/L

including one blank were used.

Data acquisition and analysis were performed using the

software Analyst 1.7.1 and Sciex OS- Q 1.4.1.20719 from

AB Sciex.

Results anddiscussion

Preparation ofDCF‑coupled beads

Superparamagnetic amino-functionalized microparticles

(BioMag® Plus) were coupled with DCF after activation

of its carboxyl function via reaction with DCC and NHS.

The DCF active ester readily reacts with amino functions on

the particle surface, anchoring the DCF moieties via amide

bonds. In a first binding test however, the so-prepared beads

showed a very high background signal that could be ascribed

to non-specific binding (NSB) of the antibody to excess free

amino groups on the bead surface. The same was observed

for the untreated particles.

In order to reduce NSB, amino functions were blocked

by reaction with cyclic anhydrides, namely succinic anhy-

dride (SA) or glutaric anhydride (GA). Binding tests with

two different monoclonal anti-DCF antibodies (F01G21 and

SK60-2E4) revealed that NSB was successfully blocked

and binding of the antibodies to the DCF moieties was not

impaired (Fig.S1).

Interestingly, the preparation of the beads had to be per-

formed in a one-pot reaction with both DCF active ester and

anhydride added at once (Fig.S2). Sequential reaction with

DCF active ester and anhydride yielded beads that gave high

NSB indicating that only the component added first is react-

ing. Accordingly, addition of anhydride before DCF active

ester led to formation of beads which showed no binding of

anti-DCF antibodies.

Besides that, incorporation of the

C6 spacer 6-aminohexa-

noic acid (Ahx) between DCF and the bead surface (S0 and

Fig.S3) was tested as well but appeared to be unsuitable

as binding of the anti-DCF antibodies was too strong and

could only be inhibited by higher DCF concentrations for the

antibody F01G21 (Fig.S4) or not inhibited by up to 10mg/L

DCF for SK60-2E4. On the other hand, the antibody SK60-

2E4 showed consistently higher IC50 values for DCF beads

(Fig.S5) showing the lower affinity of this antibody. Moreo-

ver, SK60-2E4 showed binding to beads that were coupled

with Boc-protected Ahx which could be inhibited by high

DCF concentrations (Fig.S6) indicating lower specificity of

that antibody. Against this backdrop, the following investiga-

tions were carried out with the antibody F01G21 only.

In a next step, the ratio of DCF active ester and anhy-

dride added to the bead suspension was optimized to achieve

the lowest limit of detection (LOD) in binding of dissolved

DCF (from samples). It was found that lower amounts of

DCF active ester lead to more reproducible results regard-

ing maximum O.D. values after binding of the antibodies.

Furthermore, a large excess of anhydride (50–100-fold) over

DCF active ester yielded calibration curves with the lowest

IC50 values at reasonable binding intensities. Comparing

both anhydrides, beads that were blocked with GA yielded

slightly lower IC50 values in calibration curves than those

blocked with SA (Fig.2a). Moreover, the advantage of using

a blocking reagent as additional parameter for tuning the

binding properties of the antibody could be demonstrated by

coupling DCF to a different brand of beads (Dynabeads™

M-270 Amine). These did not require additional blocking as

they did not show any NSB of the antibody. In fact, applying

a blocking reagent together with DCF active ester prevented

binding of anti-DCF antibodies to the beads completely. The

unblocked beads, however, allowed binding but calibration

curves obtained with these beads showed a significantly

higher IC50 value than BioMag® Plus beads blocked with

GA (Fig.2b).

Finally, the optimized coupling ratios for reproducible

preparation of the DCF-coupled beads were determined to

be 0.5µmol DCF active ester and 50µmol GA per 100µL of

bead suspension. Beads produced in this manner were used

to set up the competitive immunoassay for determination of

DCF in water samples.

Building thecompetitive immunoassay

Before setting up and optimizing the assay with the antibody

F01G21, the recognition element was labeled with horse-

radish peroxidase (HRP) using a commercial labeling kit.

With an achieved coupling ratio of 2.65, the sensitivity of

the assay could be increased drastically with lower amounts

of antibody needed to obtain high signal intensities. Besides,

the assay could be sped up as the additional incubation with

a secondary antibody as well as the associated washing step

could be omitted.

Assay conditions were optimized regarding the assay

buffer, incubation time, amount of bead suspension, and

antibody dilution. Regarding the assay buffer, it was found

that a Tris-based buffer with EDTA at pH7.6 was most suit-

able. While a change in buffer composition and pH did not

lead to evident shifts of the calibration curve in terms of

the IC50, the ratios of minimum and maximum O.D. varied

distinctly with the broadest range observed for Tris–EDTA

at pH 7.6 (Fig.S7a). Buffers PBS (pH7.6) and Tris–EDTA

1567A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac

1 3

(pH8.5) gave the lowest background signal but also yielded

very low maximum signal intensities, requiring more beads

and/or antibody to be increased. Both would make the assay

less cost-effective. A lower pH of 6.5 with PBS as a buffer

did not seem reasonable as here the background signal was

the highest of all tested buffers with the signal maximum

well below that of Tris–EDTA (pH 7.6). For the purpose

of analyzing water samples, the use of Tris–EDTA (pH

7.6) appeared further useful since the contained EDTA can

reduce the impact on antibody binding by chaotropic ions

like Ca2+ and Mg2+ which may be contained in higher con-

centrations in the samples.

The incubation time of the antibody with the sample and

beads was found to be sufficient to reach a desired maximum

O.D. of 1 ± 0.1 reproducibly after 20min (Fig.S7b). Longer

incubation times not only further increased the maximum

intensity but also led to higher background signals from

NSB. Therefore, and in the interest of short analysis times,

incubation was stopped after 20min, and beads were washed

to remove unbound antibody.

The antibody itself was diluted to a concentration of

9.3µg/L which yielded reasonable signal intensities at suf-

ficiently low IC50 values with an amount of 150µL of bead

suspension per plate which corresponds to approximately 1.5

µL of bead suspension per well. All steps of the immunoas-

say procedure are illustrated in Fig.3.

Under these optimized conditions, the measurement range

of the assay was determined according to the rules to set up

a precision profile described by Ekins [47] (Fig.4). Corre-

spondingly, DCF concentrations can be determined with an

error of concentration below 30% in a range from 400ng/L

to 300µg/L. Compared to the previously reported ELISA

based on the same antibody [24, 25], this represents a clear

improvement (measurement range: 3 – 150µg/L). With the

LOD significantly reduced and the analytical range broad-

ened, the newly developed assay is more versatile and can be

used not only for wastewater analysis but also for surface and

drinking water analysis as shown below. For the latter, the

German Environment Agency (UBA) suggests limit values

for DCF of 1.75µg/L (guidance value) and 20µg/L (tech-

nical action value) [48]. Both these values lie well within

the range of this assay. Therefore, it could serve as a quick

method to estimate the safety of drinking water. Moreover,

with a time of analysis of around 45min accompanied by

the high sample throughput, the magnetic bead-based assay

provides analytical results much faster than HPLC–MS and

conventional immunoassays like ELISA.

Analysis ofwater samples

In order to demonstrate the practicability of the assay for

analyzing the concentration of diclofenac in water, six differ-

ent water samples were taken and analyzed directly as well

as at three different spiking levels (24 samples in total). The

results were confirmed by LC–MS/MS analysis (TableS1).

Comparison of DCF concentrations determined by

MBBA and LC–MS/MS showed good correlation over the

covered concentration range from 500ng/L to 100µg/L

with few larger deviations occurring at elevated concentra-

tion values (Fig.5, Fig.S8, and TableS4). No false nega-

tive results were observed demonstrating that contamina-

tion of water with DCF could be detected reliably. On the

other hand, only one false positive result occurred within

the measurement range of the assay (P9) and another one

0.001 0.01 0.1 110100 1000 10000

0.0

0.2

0.4

0.6

0.8

1.0

BioMag Plus (blocked with GA)

Dynabeads (unblocked)

)mn026-mn054(.D.O

/ (µg/L)

IC50 = 3.1 µg/L

IC50 = 13 µg/L

0.001 0.010.1 110100 1000 10000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

glutaric anhydride

succinic anhydride

)mn026-mn054(.D.O

/ (µg/L)

IC50 = 9.4 µg/L

IC50 = 11 µg/L

Fig. 2 Calibration curves obtained with differently prepared beads

3, error bars represent single standard deviation). a Comparison

of the two blocking reagents GA and SA applied together with DCF

active ester on BioMag Plus® particles. b Influence of blocking com-

paring BioMag Plus® particles blocked with GA after further optimi-

zation and unblocked Dynabeads™

1568 Ecke A. et al.

1 3

Fig. 3 Schematic illustration of each assay step and the respec-

tive time frame. a Water sample containing DCF (green triangles)

is added to the suspension of DCF-coupled magnetic beads in assay

buffer. b HRP-labeled anti-DCF antibody is added and incubated with

the sample and beads for competitive binding for 20min. c Wash-

ing is performed by holding the beads with a magnet, removing the

supernatant, and adding washing buffer (repeated twice). d Substrate

solution containing TMB and hydrogen peroxide is added and incu-

bated with the particles for 15min while blue color develops upon

substrate oxidation. e The oxidation reaction is stopped by addition

of sulfuric acid with the color of the solution changing to yellow,

and beads are immobilized on the side of the well for the following f

absorption measurement in a spectrophotometer

0.001 0.010.1 110100 1000 10000

0.0

0.2

0.4

0.6

0.8

1.0

Calibration Curve

Precision Profile

/ (µg/L)

)mn026-mn054(.D.O

100

Error of Concentration / %

Fig. 4 Calibration curve of the optimized assay (orange) and preci-

sion profile (purple); n = 6; error bars represent single standard devi-

ation. The arrows indicate lower and upper limits of detection, and

thus the measurement range of the assay (400ng/L–300µg/L)

020406080100 120

100

120

012345

Concentration(MBBA)/(µg/L)

Concentration (LC-MS/MS) / (µg/L)

Fig. 5 Correlation of DCF concentrations determined by MBBA

(n = 3) and LC–MS/MS analysis (n = 4); error bars represent single

standard deviation (regression line parameters: slope m = 0.98 ± 0.03,

R2 = 0.976)

1569A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac

1 3

outside of the measurement range (P17) with a determined

concentration (203ng/L) below the actual LOD of the assay.

Apart from that, the MBBA was able to detect contamination

of the unspiked surface water sample (Teltowkanal) with

approx. 500ng/L DCF which was confirmed by LC–MS/

MS. In accordance with this, higher concentrations were

found for all three corresponding spiked surface water sam-

ples by both methods. Compared with the results of earlier

analyses of the same water in 2016 (DCF concentrations:

2.1 and 1.9µg/L) [21], the concentration determined here

appears significantly lower but may be dependent on the

sampling site. It is known that an inlet for treated wastewater

is further downstream from the sampling site so that higher

concentrations of DCF should be found there.

In order to evaluate the accuracy of the assay, recovery

rates were determined with respect to LC–MS/MS reference

measurements. In four different analyses of the same 24

samples, mean recovery rates were found in a range from

96 to 139% with overestimations at low concentration levels

shifting the data to higher values (Fig.6a, TablesS2–S6).

Regarding precision, the mean intra-assay variations

of the same four sample analyses ranged from 13 to 25%

(Fig.6b, TablesS2–S6). Higher relative variations were

found mainly for low concentration samples. As expected,

inter-assay variances were slightly higher with a mean CV

of 34% where single false-positive results of blank samples

stretched the data range to higher values. Overall, accuracy

and precision of the assay appear reasonable for quick esti-

mations of the DCF concentrations in the various tested

water samples with the main strengths of the method lying in

the high sample throughput, the broad measurement range,

short analysis times, cost-effectiveness, and the low equip-

ment expenditure.

Conclusion

The developed MBBA proved to be a fast and reliable

method to determine the DCF concentration in water sam-

ples with an LOD of 400ng/L. Due to the wide meas-

urement range and short analysis time, the MBBA pos-

sesses distinct advantages over other immunoassays such

as ELISA, which is more time-consuming and in case

of DCF less sensitive using the same antibody [24, 25].

This might be the case for other anti-DCF antibodies as

well and appears worth investigating [22, 23]. In terms

of accuracy and precision, the assay shows satisfactory

mean recovery rates of 100–140% in relation to refer-

ence analysis by LC–MS/MS, and typical coefficients of

variation of 10–25% (intra-assay) and ~ 30% (inter-assay).

The MBBA only requires manual washing steps, using a

pipette, and therefore does not require a microplate washer

which makes its implementation easier and enhances its

field portability. With this, the assay provides a quick and

easy method to assess contamination of water and in this

context the safety of its use as a source of drinking water.

Prospectively, the assay principle can be transferred to

other analytes with the same improvement in terms of analy-

sis time and sensitivity as for DCF, having the potential to

replace ELISA as the standard technique in immunoanalysis.

Incorporation of the magnetic beads into a lateral flow sys-

tem appears feasible as well and represents another future

application of our magnetic beads. As shown previously,

changing the detection mode from optical to electrochemical

detection is also possible which allows for further miniaturi-

zation of the system and mobile testing [49]. Beyond that,

the use of magnetic beads will also enable automation and

implementation of the assay into autonomous sensors for

Intra-Assay 1Intra-Assay 2Intra-Assay 3Intra-Assay 4Inter-Assay

100

CV / %

25%~75%

1%~99%

Median Line

Mean

Outliers

1234Mean

100

120

140

160

180

200

220

240

etaRyrevoceR / %

25%~75%

1%~99%

Median Line

Mean

Outliers

Fig. 6 a Recovery rates (without blanks, n = 19) in four independ-

ent analyses and mean of all four measurements for water samples

compared to LC–MS/MS reference. b Intra-assay variations of four

independent sample analyses in triplicate each (n = 3, m = 24) and

inter-assay variation of the mean of these four measurements (n = 4,

m = 24)

1570 Ecke A. et al.

1 3

possible on-site analysis. Our efforts in this direction focus

on developing an integrated diagnosis system for pharma-

ceutical contaminants directly in water supply pipes.

Supplementary Information The online version contains supplemen-

tary material available at https:// doi. org/ 10. 1007/ s00216- 021- 03778-7.

Acknowledgements We thank the Indo-German Science and Technol-

ogy Centre (IGSTC) and Federal Ministry for Education and Research

(BMBF) for funding of the associated project IDC-Water (Integrated

Diagnostics of Contaminants in Water supply and management sys-

tems)as well as the project partners for the successful collaboration.

We further thank Soraya Höfs (BAM) for support in water sampling

and Valerie Jaut (BAM) for lab assistance.

Author contribution Conceptualization: Alexander Ecke. Method-

ology: Alexander Ecke. Formal analysis: Alexander Ecke and Tanja

Westphalen. Investigation: Alexander Ecke, Tanja Westphalen and

Jane Hornung. Validation: Alexander Ecke and Tanja Westphalen.

Resources: Michael Voetz and Rudolf J. Schneider. Data curation:

Alexander Ecke. Visualization: Alexander Ecke. Writing–original

draft preparation: Alexander Ecke. Writing–review and editing: Tanja

Westphalen, Jane Hornung, Michael Voetz and Rudolf J. Schneider.

Supervision: Michael Voetz and Rudolf J. Schneider. Project adminis-

tration: Michael Voetz and Rudolf J. Schneider. Funding acquisition:

Michael Voetz and Rudolf J. Schneider. All the authors have read and

agreed to the published version of the manuscript.

Funding Open Access funding enabled and organized by Projekt

DEAL. This work contributes to the project IDC-Water co-funded by

the Indo-German Science and Technology Centre (IGSTC) and Fed-

eral Ministry for Education and Research (BMBF), grant number FKZ

01DQ18003A.

Data availability Supplementary Material is available…

Code availability Not applicable.

Declarations

Conflict of interest The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attri-

bution 4.0 International License, which permits use, sharing, adapta-

tion, distribution and reproduction in any medium or format, as long

as you give appropriate credit to the original author(s) and the source,

provide a link to the Creative Commons licence, and indicate if changes

were made. The images or other third party material in this article are

included in the article’s Creative Commons licence, unless indicated

otherwise in a credit line to the material. If material is not included in

the article’s Creative Commons licence and your intended use is not

permitted by statutory regulation or exceeds the permitted use, you will

need to obtain permission directly from the copyright holder. To view a

copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

References

1. Kantor TG. Use of diclofenac in analgesia. Am J Med. 1986;80(4,

Supplement 2):64–9. https:// doi. org/ 10. 1016/ 0002- 9343(86)

90083-5.

2. Altman R, Bosch B, Brune K, Patrignani P, Young C. Advances

in NSAID development: evolution of diclofenac products using

pharmaceutical technology. Drugs. 2015;75(8):859–77. https://

doi. org/ 10. 1007/ s40265- 015- 0392-z.

3. McGettigan P, Henry D. Use of non-steroidal anti-inflammatory

drugs that elevate cardiovascular risk: an examination of sales

and essential medicines lists in low-, middle-, and high-income

countries. PLOS Med. 2013;10(2):e1001388. https:// doi. org/ 10.

1371/ journ al. pmed. 10013 88.

4. Bruun Kristensen K, Karlstad Ø, Martikainen JE, Pottegård A,

Wastesson JW, Zoega H, Schmidt M. Nonaspirin nonsteroidal

antiinflammatory drug use in the Nordic countries from a cardio-

vascular risk perspective, 2000–2016: a drug utilization study.

Pharmacotherapy. 2019;39(2):150–60. https:// doi. org/ 10. 1002/

phar. 2217.

5. Vieno N, Sillanpää M. Fate of diclofenac in municipal wastewater

treatment plant — a review. Environ Int. 2014;69:28–39. https://

doi. org/ 10. 1016/j. envint. 2014. 03. 021.

6. Zhang Y, Geißen S-U, Gal C. Carbamazepine and diclofenac:

removal in wastewater treatment plants and occurrence in water

bodies. Chemosphere. 2008;73(8):1151–61. https:// doi. org/ 10.

1016/j. chemo sphere. 2008. 07. 086.

7. Schwaiger J, Ferling H, Mallow U, Wintermayr H, Negele

RD. Toxic effects of the non-steroidal anti-inflammatory drug

diclofenac: part I: histopathological alterations and bioaccumula-

tion in rainbow trout. Aquat Toxicol. 2004;68(2):141–50. https://

doi. org/ 10. 1016/j. aquat ox. 2004. 03. 014.

8. Triebskorn R, Casper H, Heyd A, Eikemper R, Köhler HR,

Schwaiger J. Toxic effects of the non-steroidal anti-inflamma-

tory drug diclofenac: part II. Cytological effects in liver, kid-

ney, gills and intestine of rainbow trout (Oncorhynchus mykiss).

Aquat Toxicol. 2004;68(2):151–66. https:// doi. org/ 10. 1016/j.

aquat ox. 2004. 03. 015.

9. Oaks JL, Gilbert M, Virani MZ, Watson RT, Meteyer CU,

Rideout BA, Shivaprasad HL, Ahmed S, Chaudhry MJI,

Arshad M, Mahmood S, Ali A, Khan AA. Diclofenac residues

as the cause of vulture population decline in Pakistan. Nature.

2004;427(6975):630–3. https:// doi. org/ 10. 1038/ natur e02317.

10. Saini M, Taggart MA, Knopp D, Upreti S, Swarup D, Das

A, Gupta PK, Niessner R, Prakash V, Mateo R, Cuthbert RJ.

Detecting diclofenac in livestock carcasses in India with an

ELISA: a tool to prevent widespread vulture poisoning. Environ

Pollut. 2012;160:11–6. https:// doi. org/ 10. 1016/j. envpol. 2011.

09. 011.

11. Bonnefille B, Gomez E, Courant F, Escande A, Fenet H.

Diclofenac in the marine environment: a review of its occurrence

and effects. Mar Pollut Bull. 2018;131:496–506. https:// doi. org/

10. 1016/j. marpo lbul. 2018. 04. 053.

12. Sathishkumar P, Meena RAA, Palanisami T, Ashokkumar V, Pal-

vannan T, Gu FL. Occurrence, interactive effects and ecological

risk of diclofenac in environmental compartments and biota - a

review. Sci Total Environ. 2020;698:134057. https:// doi. org/ 10.

1016/j. scito tenv. 2019. 134057.

13. Commission Implementing Decision (EU) 2015/495 of 20 March

2015 establishing a watch list for Union-wide monitoring in the

field of water policy pursuant to Directive 2008/105/EC of the

European Parliament and of the Council. Official Journal of the

European Union2015. p. 40-2.

14. Proposal for a Directive of the European Parliament and of the

Council amending Directives 2000/60/EC and 2008/105/EC as

regards priority substances in the field of water policy. European

Commission; 2012. p. COM/2011/0876 final - 2011/0429 (COD).

15. Huber C, Preis M, Harvey PJ, Grosse S, Letzel T, Schröder

P. Emerging pollutants and plants – metabolic activation of

diclofenac by peroxidases. Chemosphere. 2016;146:435–41.

https:// doi. org/ 10. 1016/j. chemo sphere. 2015. 12. 059.

1571A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac

1 3

16. Jewell KS, Falås P, Wick A, Joss A, Ternes TA. Transformation

of diclofenac in hybrid biofilm–activated sludge processes. Water

Res. 2016;105:559–67. https:// doi. org/ 10. 1016/j. watres. 2016. 08.

002.

17. Rigobello ES, Di Bernardo DA, Di Bernardo L, Vieira EM.

Removal of diclofenac by conventional drinking water treatment

processes and granular activated carbon filtration. Chemosphere.

2013;92(2):184–91. https:// doi. org/ 10. 1016/j. chemo sphere. 2013.

03. 010.

18. Langenhoff A, Inderfurth N, Veuskens T, Schraa G, Blokland M,

Kujawa-Roeleveld K, Rijnaarts H. Microbial removal of the phar-

maceutical compounds ibuprofen and diclofenac from wastewa-

ter. BioMed Res Int. 2013;2013:325806. https:// doi. org/ 10. 1155/

2013/ 325806.

19. Rosset M, Weidlich Sfreddo L, Navarro Hidalgo GE, Perez-

Lopez OW, Amaral FL. Adsorbents derived from hydrotalcites

for the removal of diclofenac in wastewater. Appl Clay Sci.

2019;175:150–8. https:// doi. org/ 10. 1016/j. clay. 2019. 04. 014.

20 Angosto JM, Roca MJ, Fernández-López JA. Removal of

diclofenac in wastewater using biosorption and advanced oxida-

tion techniques: comparative results. Water. 2020;12(12):3567.

https:// doi. org/ 10. 3390/ w1212 3567.

21. Schmidt S, Hoffmann H, Garbe L-A, Schneider RJ. Liquid chro-

matography–tandem mass spectrometry detection of diclofenac

and related compounds in water samples. J Chromatogr A.

2018;1538:112–6. https:// doi. org/ 10. 1016/j. c hr oma. 2018. 01. 037.

22. Deng A, Himmelsbach M, Zhu Q-Z, Frey S, Sengl M, Buchberger

W, Niessner R, Knopp D. Residue analysis of the pharmaceutical

diclofenac in different water types using ELISA and GC−MS.

Environ Sci Technol. 2003;37(15):3422–9. https:// doi. org/ 10.

1021/ es034 1945.

23. Huebner M, Weber E, Niessner R, Boujday S, Knopp D. Rapid

analysis of diclofenac in freshwater and wastewater by a mon-

oclonal antibody-based highly sensitive ELISA. Anal Bio-

anal Chem. 2015;407(29):8873–82. https:// doi. org/ 10. 1007/

s00216- 015- 9048-9.

24. Harrer A, Lang R, Grims R, Braitsch M, Hawranek T, Aberer

W, Vogel L, Schmid W, Ferreira F, Himly M. Diclofenac hyper-

sensitivity: antibody responses to the parent drug and relevant

metabolites. PLoS ONE. 2010;5(10):e13707. https:// doi. org/ 10.

1371/ journ al. pone. 00137 07.

25. Schmidt S. Hapten synthesis for the antibody-based detection of

diclofenac in water samples [Dissertation]: Technische Universität

Berlin; 2019.

26. Hlaváček A, Peterek M, Farka Z, Mickert MJ, Prechtl L, Knopp D,

Gorris HH. Rapid single-step upconversion-linked immunosorb-

ent assay for diclofenac. Microchim Acta. 2017;184(10):4159–65.

https:// doi. org/ 10. 1007/ s00604- 017- 2456-0.

27. Raysyan A, Moerer R, Coesfeld B, Eremin SA, Schneider RJ.

Fluorescence polarization immunoassay for the determination of

diclofenac in wastewater. Anal Bioanal Chem. 2021;413(4):999–

1007. https:// doi. org/ 10. 1007/ s00216- 020- 03058-w.

28. Carl P, Sarma D, Gregório BJR, Hoffmann K, Lehmann A, Rurack

K, Schneider RJ. Wash-free multiplexed mix-and-read suspen-

sion array fluorescence immunoassay for anthropogenic markers

in wastewater. Anal Chem. 2019;91(20):12988–96. https:// doi. or g/

10. 1021/ acs. analc hem. 9b030 40.

29. Nguyen TTK, Vu TT, Anquetin G, Tran HV, Reisberg S, Noël V,

Mattana G, Nguyen QV, Dai Lam T, Pham MC, Piro B. Enzyme-

less electrochemical displacement heterogeneous immunosensor

for diclofenac detection. Biosens Bioelectron. 2017;97:246–52.

https:// doi. org/ 10. 1016/j. bios. 2017. 06. 010.

30. Chen W, Zhu Q, Tang Q, Zhao K, Deng A, Li J. Ultrasensitive

detection of diclofenac based on electrochemiluminescent immu-

nosensor with multiple signal amplification strategy of palladium

attached graphene oxide as bioprobes and ceria doped zinc oxide

as substrates. Sensor Actuat B-Chem. 2018;268:411–20. https://

doi. org/ 10. 1016/j. snb. 2018. 04. 106.

31. Hu L, Zheng J, Zhao K, Deng A, Li J. An ultrasensitive elec-

trochemiluminescent immunosensor based on graphene oxide

coupled graphite-like carbon nitride and multiwalled carbon

nanotubes-gold for the detection of diclofenac. Biosens Bioel-

ectron. 2018;101:260–7. https:// doi. or g/ 10. 1016/j. bios. 2017. 10.

043.

32. Shi J, Xu M, Tang Q, Zhao K, Deng A, Li J. Highly sensitive

determination of diclofenac based on resin beads and a novel

polyclonal antibody by using flow injection chemiluminescence

competitive immunoassay. Spectrochim Acta A. 2018;191:1–7.

https:// doi. org/ 10. 1016/j. saa. 2017. 09. 068.

33. Kaewwonglom N, Oliver M, Cocovi-Solberg DJ, Zirngibl K,

Knopp D, Jakmunee J, Miró M. Reliable sensing platform for

plasmonic enzyme-linked immunosorbent assays based on auto-

matic flow-based methodology. Anal Chem. 2019;91(20):13260–

7. https:// doi. org/ 10. 1021/ acs. analc hem. 9b038 55.

34. Steinke N, Döring S, Wuchrer R, Kroh C, Gerlach G, Härtling T.

Plasmonic sensor for on-site detection of diclofenac molecules.

Sensor Actuat B-Chem. 2019;288:594–600. https:// doi. org/ 10.

1016/j. snb. 2019. 02. 069.

35. Wang C, Jiang T, Zhao K, Deng A, Li J. A novel electrochemi-

luminescent immunoassay for diclofenac using conductive poly-

mer functionalized graphene oxide as labels and gold nanorods

as signal enhancers. Talanta. 2019;193:184–91. https:// doi. org/

10. 1016/j. talan ta. 2018. 09. 103.

36. Choi J-W, Oh KW, Thomas JH, Heineman WR, Halsall HB,

Nevin JH, Helmicki AJ, Henderson HT, Ahn CH. An integrated

microfluidic biochemical detection system for protein analy-

sis with magnetic bead-based sampling capabilities. Lab Chip.

2002;2(1):27–30. https:// doi. org/ 10. 1039/ B1075 40N.

37. Hervás M, López MÁ, Escarpa A. Simplified calibration and anal-

ysis on screen-printed disposable platforms for electrochemical

magnetic bead-based inmunosensing of zearalenone in baby food

samples. Biosens Bioelectron. 2010;25(7):1755–60. https:// doi.

org/ 10. 1016/j. bios. 2009. 12. 031.

38. Kwon Y, Hara CA, Knize MG, Hwang MH, Venkateswaran KS,

Wheeler EK, Bell PM, Renzi RF, Fruetel JA, Bailey CG. Mag-

netic bead based immunoassay for autonomous detection of tox-

ins. Anal Chem. 2008;80(22):8416–23. https:// doi. org/ 10. 1021/

ac801 0044.

39. Hendrickson OD, Chertovich JO, Zherdev AV, Sveshnikov PG,

Dzantiev BB. Ultrasensitive magnetic ELISA of zearalenone with

pre-concentration and chemiluminescent detection. Food Control.

2018;84:330–8. https:// doi. org/ 10. 1016/j. foodc ont. 2017. 08. 008.

40. Kantiani L, Farré M, Asperger D, Rubio F, González S, López

de Alda MJ, Petrović M, Shelver WL, Barceló D. Triclosan and

methyl-triclosan monitoring study in the northeast of Spain using

a magnetic particle enzyme immunoassay and confirmatory

analysis by gas chromatography–mass spectrometry. J Hydrol.

2008;361(1):1–9. https:// doi. org/ 10. 1016/j. jhydr ol. 2008. 07. 016.

41. Wang Y-K, Wang Y-C, Wang H, Ji W-H, Sun J-H, Yan Y-X. An

immunomagnetic-bead-based enzyme-linked immunosorbent

assay for sensitive quantification of fumonisin B1. Food Control.

2014;40:41–5. https:// doi. org/ 10. 1016/j. foodc ont. 2013. 11. 025.

42. Zhao F, Shen Q, Wang H, Han X, Yang Z. Development of a

rapid magnetic bead-based immunoassay for sensitive detection

of zearalenone. Food Control. 2017;79:227–33. https:// doi. org/

10. 1016/j. foodc ont. 2017. 03. 051.

43. Deng Q, Qiu M, Wang Y, Lv P, Wu C, Sun L, Ye R, Xu D, Liu Y,

Gooneratne R. A sensitive and validated immunomagnetic-bead

based enzyme-linked immunosorbent assay for analyzing total T-2

(free and modified) toxins in shrimp tissues. Ecotox Environ Safe.

2017;142:441–7. https:// doi. org/ 10. 1016/j. ecoenv. 2017. 04. 037.

1572 Ecke A. et al.

1 3

44. Xin T-B, Wang X, Jin H, Liang S-X, Lin J-M, Li Z-J. Devel-

opment of magnetic particle-based chemiluminescence enzyme

immunoassay for the detection of 17β-estradiol in environmental

water. Appl Biochem Biotech. 2009;158(3):582–94. https:// doi.

org/ 10. 1007/ s12010- 008- 8356-3.

45. Huergo LF, Selim KA, Conzentino MS, Gerhardt ECM, Santos

ARS, Wagner B, Alford JT, Deobald N, Pedrosa FO, de Souza

EM, Nogueira MB, Raboni SM, Souto D, Rego FGM, Zanette DL,

Aoki MN, Nardin JM, Fornazari B, Morales HMP, Borges VA,

Nelde A, Walz JS, Becker M, Schneiderhan-Marra N, Rothbauer

U, Reis RA, Forchhammer K. Magnetic bead-based immunoassay

allows rapid, inexpensive, and quantitative detection of human

SARS-CoV-2 antibodies. ACS Sens. 2021;6(3):703–8. https:// doi.

org/ 10. 1021/ acsse nsors. 0c025 44.

46. Wilson MB, Nakane PP. Recent developments in the periodate

method of conjugating horseradish peroxidase (HRP) to antibod-

ies. In: Knapp W, Holubar K, Wick G, editors. Immunofluores-

cence and related staining techniques. Amsterdam: Elsevier/North

Holland Biomedical; 1978. p. 215–24.

47. Ekins RP. The precision profile: its use in assay design, assess-

ment and quality control. In: Hunter WM, Corrie JET, editors.

Immunoassays for clinical chemistry. 2nd ed. Edinburgh: Church-

ill Livingstone; 1983. p. 76–105.

48 Dieter HH. Grenzwerte, Leitwerte, Orientierungswerte, Maßnah-

menwerte

- Aktuelle Definitionen und Höchstwerte. Dessau-

Roßlau: Umweltbundesamt; 2011.

49. Höfs S, Hülagü D, Bennet F, Carl P, Flemig S, Schmid T, Schenk

JA, Hodoroaba V-D, Schneider RJ. Electrochemical immunomag-

netic ochratoxin A sensing: steps forward in the application of

3,3’,5,5’-tetramethylbenzidine in amperometric assays. ChemE-

lectroChem. 2021;8(13):2597–606. https:// doi. org/ 10. 1002/ celc.

20210 0446.

Publisher’s note Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations.

1573A rapid magnetic bead-based immunoassay for sensitive determination of diclofenac