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Article
A competitive Upconversion-Linked Immunosorbent Assay (ULISA) for the rapid and sensitive detection of diclofenac Antonín Hlavá#ek, Zden#k Farka, Maria Hübner, Veronika Hor#áková, Daniel Nemecek, Reinhard Niessner, Petr Skládal, Dietmar Knopp, and Hans H Gorris Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01083 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016
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Analytical Chemistry
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A competitive Upconversion-Linked Immunosorbent Assay
2
(ULISA) for the rapid and sensitive detection of diclofenac
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Antonín Hlaváček†,‡,§, Zdeněk Farka†,‡, Maria Hübnerǁ, Veronika Horňáková‡, Daniel
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Němeček‡, Reinhard Niessnerǁ, Petr Skládal‡, Dietmar Knoppǁ, Hans H. Gorris†*
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†
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, 93040 Regensburg, Germany ‡
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CEITEC - Central European Institute of Technology, Masaryk University, Brno 625 00, Czech Republic
11
§
12 13
ǁ
Institute of Analytical Chemistry AS CR, v. v. i., Brno 602 00, Czech Republic
Chair for Analytical Chemistry and Institute of Hydrochemistry, Technical University of Munich, 81377 Munich, Germany
14 15 16 17 18 19 20 21 22
*Corresponding author
23
Hans H. Gorris, PhD
24 25 26 27
Institute of Analytical Chemistry, Chemo- and Biosensors University of Regensburg Universitätsstr. 31 93040 Regensburg
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Germany
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Tel.: +49-941-943-4015
30
Fax: +49-941-943-4064
31
e-mail:
[email protected] 1 ACS Paragon Plus Environment
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Abstract
33
Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-
34
infrared excitation and thus avoid optical background interference. We have exploited this
35
unique photophysical feature to establish a sensitive competitive immunoassay for the
36
detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called
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upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of
38
the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter)
39
exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody.
40
We investigated the structure and monodispersity of the nanoconjugates in detail. Using a
41
highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of
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0.05 ng DCF per mL. This performance comes close to a conventional ELISA without the
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need for an enzyme-mediated signal amplification step. The ULISA was further employed for
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analyzing drinking and surface water samples and the results were consistent with a
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conventional ELISA as well as LC-MS.
46 47
Introduction
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The enzyme-linked immunosorbent assay (ELISA) is a cost efficient tool for the specific and
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highly sensitive detection of many toxic analytes in food and environmental samples as well
50
as clinical diagnosis. There are, however, some disadvantages of a classic ELISA such as an
51
inherent instability of enzymes and time consuming signal development. Consequently, many
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research efforts have been made to replace the enzymes by using nanoparticles (NPs) as
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signal amplifiers, e.g. fluorescent dye-doped polymer or silica NPs,1 metal NPs,2 magnetic
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NPs,3
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(UCNPs) have been used as a new generation of luminescent labels for sensitive
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immunochemical detection. UCNPs are lanthanide-doped nanocrystals that can be excited by
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near-infrared light and emit light of shorter wavelengths (anti-Stokes emission),6,7 which
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strongly reduces autofluorescence and light scattering. Further advantages of UCNPs include
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(A) a very high photostability, (B) large anti-Stokes shifts allowing for an excellent separation
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of excitation and detection channels, and (C) multiple and narrow emission bands that can be
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tuned individually for the multiplexed detection of analytes.8-10
catalytic NPs4 or quantum dots.5 Recently, photon-upconverting nanoparticles
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These distinct photophysical features of UCNPs have been used for the design of
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heterogeneous microtiter plate immunoassays, e.g. for the detection of prostate-specific 2 ACS Paragon Plus Environment
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antigen (limit of detection, LOD: 0.15 ng mL-1 / 6 pM)11 or human chorionic gonadotropin
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(LOD: 3.8 ng mL-1 / 200 pM).12 The advantages of UCNPs in lateral flow assays e.g. for the
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detection of worm parasite antigens (LOD: 0.01 ng mL-1 / 0.1 pM)13 have also been well
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documented. There have been a few reports on the use of homogeneous competitive
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immunoassays for the detection of small molecules such as estradiol (LOD: ~0.1 ng mL-1 /
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400 pM)14 and folate (LOD: 0.4 ng mL-1 / 1000 pM)15 in blood and a bead-based
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immunoassay for the detection of mycotoxins in food samples (LOD: ~0.01 ng mL-1 / 50
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pM).16
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The development and widespread availability of more sensitive analytical techniques has
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resulted in an increasing number of pharmaceuticals that can be detected in the environment
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after medical or veterinary use.17,18 Diclofenac (2-[2-(2,6-dichlorophenyl) aminophenyl]
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ethanoic acid; DCF) is a widely used non-steroidal anti-inflammatory drug (NSAID). In the
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Indian subcontinent, the widespread use of DCF for veterinary treatment of cattle since the
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1990s has led to a precipitous decline of the indigenous vulture population because DCF leads
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to renal failure in vultures that feed on contaminated carcasses.19 In Europe, DCF belongs to
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the most frequently detected pharmaceuticals in the water-cycle because it is not easily
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degraded when passing through sewage treatment plants. DCF has been detected in low µg
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L−1 amounts in wastewater effluents and also in ng L-1 amounts in surface waters,20
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groundwater and drinking water.21 Very low amounts of DCF can be detected by LC-TOF-
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MS or high resolution mass spectrometers.22,23 These instrumental techniques, however, are
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expensive, time consuming, labor intensive and need trained personnel. By contrast,
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immunoassays are more suitable for on-site testing directly in the field or for the analysis of
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large numbers of samples in small laboratories.24
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Here, we have optimized the preparation of monodisperse and stable upconversion
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reporters for the sensitive detection of DCF in water samples by a competitive upconversion-
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linked immunosorbent assay (ULISA). Anti-mouse IgG antibodies were conjugated to silica-
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coated UCNPs exposing carboxyl function on the surface and the conjugates were
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characterized by gel electrophoresis.25 The competitive detection of DCF was performed
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using a monoclonal mouse anti-DCF antibody (Figure 1). This antibody was characterized in
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detail as described recently26 and showed about 10 % cross-reactivity with DCF metabolites
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such as 5-OH-DCF, 4’-OH-DCF and DCF-acyl glucuronide, but only less than 1 % with other
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structurally related non-steroidal anti-inflammatory drugs. The performance of the optimized
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ULISA was compared with a conventional ELISA as well as LC-MS. 3 ACS Paragon Plus Environment
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Figure 1. Scheme of the indirect competitive ULISA for the detection of diclofenac (DCF). (A) A microtiter plate is coated with a bovine serum albumin-DCF conjugate (BSA-DCF). (B) Dilution series of DCF are prepared in the microtiter plate followed by the addition of anti-diclofenac mouse antibody. (C) The attachment of anti-diclofenac antibody is then detected by an anti-mouse IgG-UCNP secondary antibody conjugate. The upconversion luminescence is recorded under 980 nm laser excitation.
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Experimental section
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Chemicals
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All standard chemicals and diclofenac sodium salt (D6899, purity ≥98 %) were obtained from
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Sigma-Aldrich (Steinheim, Germany). Carboxyethylsilanetriol sodium salt; 25% (w/v) in
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water was obtained from ABCR GmbH (Karlsruhe, Germany). The horseradish peroxidase-
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labeled horse anti-mouse IgG was from Axxora (Lörrach, Germany) and horse anti-mouse
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IgG was from Vector Laboratories (Burlingame, USA). The monoclonal anti-diclofenac
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antibody 12G5 was generated in mice using a DCF-thyroglobulin conjugate as described
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previously.26 An antibody stock solution of 0.45 mg mL-1 was prepared in 20 mM NaH2PO4,
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20 mM NaH2PO4, 0.1 M Tris-HCl, 0.02% NaN3, pH 7.4 and stored at 4° C. Buffers and
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solutions were prepared with ultrapure water, which was obtained by reverse osmosis with
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UV treatment (Milli-RO 5 Plus, Milli-Q185 Plus, Eschborn, Germany).
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Synthesis of carboxyl-silica-coated UCNPs
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UCNPs of 42.5 ± 4.9 nm in diameter were synthesized by high-temperature co-precipitation27
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as described in the Supporting Information. The mass concentration was determined by
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gravimetric analysis and a concentration of 1.0 mg mL-1 of UCNPs was estimated to be
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equivalent to the molar concentration of 9.8×10-9 mol L-1 (Supporting Information).
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Carboxyl-silica-coated
UCNPs
(COOH-UCNPs)
were
prepared
by
a
reverse
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microemulsion method:25 UCNPs (80 mg) were diluted in cyclohexane to a final volume of
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23 mL. This dispersion was mixed with 1800 mg of Igepal CO-520 and 100 µL of tetraethyl
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orthosilicate (TEOS) and stirred intensively for 10 min. A mixture of 55 µL 25 % (w/v)
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aqueous ammonium hydroxide and 55 µL water was added to form a microemulsion that was 4 ACS Paragon Plus Environment
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slowly stirred overnight. Then, 25 µL of TEOS were added and the microemulsion was again
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stirred for 180 min. After adding 50 µL of 25% (w/v) sodium carboxyethylsilanetriol in water,
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the microemulsion was first sonicated for 15 min and then stirred for 60 min. The COOH-
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UCNPs were extracted with 1000 µL of dimethylformamide and washed four times with 20
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mL of propan-2-ol, three times with 5000 µL of water and finally dispersed in water to yield a
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final concentration of 150 mg mL-1. The COOH-UCNPs in water were stable at 4°C for
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several months.
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Conjugation of COOH-UCNP and secondary antibody
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COOH-UCNPs were first activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
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(EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). In a typical synthesis, 0.5 mg (~5 pmol)
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of COOH-UCNPs was dispersed in water to a final volume of 200 µL. The volume of 50 µL
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of a mixture containing 2.1 µmol of EDC and 5.5 µmol of sulfo-NHS in 100 µL of 100 mM
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sodium 2-(N-morpholino)ethanesulfonate (MES) buffer, pH 6.1 was added and mixed for 30
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min. A dispersion of 100 µL activated COOH-UCNPs (1 mg mL-1 or ~10 nmol L-1) were
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mixed with 100 µL of horse anti-mouse IgG in borate buffer (100 mM sodium borate, pH
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9.0). Three IgG concentrations were employed and incubated for 90 min at room temperature:
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1) 330 nmol L-1 IgG resulting in a ratio 33 IgG molecules per UCNP (sample IgG-UCNP-
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33:1). 2) 67 nmol L-1 IgG resulting in a ratio of 7 IgG molecules per UCNP (IgG-UCNP-7:1),
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and 3) 33 nmol L-1 IgG resulting in a ratio of 3 IgG molecules per UCNP (IgG-UCNP-3:1).
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The bioconjugates were centrifuged for 10 min at 4,000 g, dispersed in UCNP assay buffer
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(50 mM Tris, 150 mM NaCl, 0.05% NaN3, 0.01% Tween 20, 0.05% bovine γ-globulin
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(BGG), 0.5% bovine serum albumin (BSA), 0.2% polyvinyl alcohol 6000 (PVA), pH 7.75)
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and sonicated for 5 min.
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Nanoparticle characterization
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Transmission electron microscopy (TEM) was performed on a Tecnai F20 FEI instrument
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(Eindhoven, The Netherlands). About 4 µL of UCNPs were deposited on a 400-mesh copper
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EM grid coated with a continuous carbon layer and negatively stained with 2% (w/v) aqueous
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solution of uranyl acetate to increase the contrast of the silica shell. The dried grids were then
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imaged at 50,000× magnification (2.21 Å pixel-1). The size of individual particles in the TEM
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images was measured by the imaging software ImageJ (http://imagej.nih.gov).28 The
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hydrodynamic diameter and zeta potential of UCNP suspensions were determined on a
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Zetasizer Nano SZ from Malvern Instruments (Malvern, UK). FT-IR spectra were recorded
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on an Alpha FTIR Spectrometer from Bruker (Billerica, USA). 5 ACS Paragon Plus Environment
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Agarose gel electrophoresis
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Following our previous work,25 COOH-UCNPs and their bioconjugates were characterized by
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agarose gel electrophoresis (0.5% w/v agarose, 45 mM Tris, 45 mM H3BO3 with pH 8.6, 15
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min at 100 V). Samples were mixed in a ratio of 10:1 with 50 % w/w glycerol and 8 µL
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aliquots were loaded onto the gel. A custom-built upconversion reader (CHAMELEON
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multilabel microplate reader, Hidex, Turku, Finland) equipped with a continuous 980 nm laser
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(4 W) was used to scan agarose gels with a spatial resolution of 0.5 mm as described earlier.29
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Conjugation of diclofenac to BSA
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BSA-DCF conjugates were prepared using either 1.5 µmol or 7.5 µmol DCF and 9.7 µmol
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sulfo-NHS added to a mixture of 400 µL MES buffer and 100 µL dimethylformamide. DCF
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was activated by the addition of 47 µmol of EDC and incubation at room temperature for 30
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min. After adding 500 µL of 0.15 µmol BSA in water and 250 µL of 50 mM aqueous Na2CO3,
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the mixture was incubated at room temperature for 4 hours and then dialyzed three times
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against 150 mL of 50 mM Na2CO3. BSA-DCF was adjusted to a concentration of 1.6 mg
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mL−1 by adding 50 mM of Na2CO3 and stored at 4 °C in presence of 0.05 % NaN3. The
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conjugate was analyzed by MALDI-TOF-MS (Bruker, Ultraflex TOF/TOF, N2-laser, 337 nm,
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positive mode).
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Water samples
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Munich tap water and two surface water samples were collected in Southern Bavaria from
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lake Wörthsee and river Würm. The fresh water samples were filtrated over a glass microfiber
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filter (GF/C, Whatman Cat. No. 1822 047) and stored at 4°C. The concentrations of Ca2+,
180
Mg2+, and dissolved organic content (DOC) as well as the conductivity and pH were
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determined (Supporting Information Table 1). ELISA and ULISA were performed with
182
undiluted and spiked samples. For LC-MS, the samples were subjected to generic solid phase
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extraction (SPE) and analyzed by an Orbitrap-based ExactiveTM Benchtop Mass Spectrometer
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(Thermo Scientific, Dreieich, Germany) as described earlier26.
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Upconversion-linked immunosorbent assay (ULISA)
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A transparent 96-well microtiter plate with high protein binding capacity (Corning,
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Wiesbaden, Germany) was coated with BSA-DCF in coating buffer (optimal concentration: 1
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µg mL-1 BSA-DCF in 50 mM NaHCO3 /Na2CO3, 0.05% NaN3, pH 9.6; 200 µL per well) at
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4° C over night. All subsequent steps were carried out at room temperature. The plate was
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washed manually four times with 250 µL of washing buffer (50 mM Na/H2PO4/HPO4, 0.01% 6 ACS Paragon Plus Environment
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Tween 20, 0.05% NaN3, pH 7.4). The free binding sites in each well were blocked with 250
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µL of 1% BSA in 50 mM Na/H2PO4/HPO4, 0.05 % NaN3, pH 7.4 for 1 h. The plate was
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washed four times with washing buffer. Either standard dilutions of DCF in double distilled
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water or environmental samples (100 µL per well) were added, immediately followed by the
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anti-DCF monoclonal mouse antibody (12G5, optimal concentration: 0.225 µg mL−1 in 100
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mM NaPO4, 300 mM NaCl, 100 µL per well) and incubated for 1 h. After four washing steps,
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the microtiter plate was incubated for one hour with 100 µL of the IgG-UCNP conjugate
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(optimal concentration: 10 µg mL−1 in 50 mM Tris, 150 mM NaCl, 0.05% NaN3, 0.01%
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Tween 20, 0.05% BGG, 0.5% BSA, 0.2% PVA, pH 7.75). After four washing steps, the
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upconversion luminescence was read out from empty wells using a custom-built upconversion
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microplate reader (CHAMELEON multilabel microplate reader, Hidex, Turku, Finland)
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equipped with a continuous 980 nm laser (4 W). A collimated laser spot of ~0.8 mm was
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focused on the bottom of the microtiter wells. Each well was scanned 100 times in a raster
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with the step size of 0.4 mm and 500 ms signal integration time. The truncated mean was
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calculated for each well after discarding the ten highest and ten lowest measurements of the
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luminescence intensity to account for local irregularities on the microtiter well surface that
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result in signal outliers.
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Enzyme-linked immunosorbent assay (ELISA)
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The ELISA was performed as described earlier.26 A transparent 96-well microtiter plate with
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high protein binding capacity (Greiner Bio-one, Frickenhausen, Germany) was coated with
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0.5 µg mL−1 of ovalbumin-DCF conjugate in coating buffer (50 mM NaHCO3/Na2CO3, 0.05%
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NaN3, pH 9.6; 200 µL per well) at 4 °C over night. All subsequent steps were carried out at
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room temperature. The plate was automatically washed with a plate washer (ELx405 Select,
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Bio-Tek Instruments, Bad Friedrichshall, Germany) four times with washing buffer (50 mM
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K/H2PO4/HPO4, 146 mM NaCl, 0.05% Tween 20, pH 7.6; PBST). The free binding sites in
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each well were blocked with 300 µL of 1% BSA in PBST for 1 h. The plate was washed four
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times with washing buffer. First, standard dilutions of DCF in double distilled water or
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environmental samples (100 µL per well) were added, immediately followed by the anti-DCF
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monoclonal mouse antibody (12G5, 0.5 µg mL−1 in PBS; 100 µL per well) and incubated for
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30 min. After four washing steps, the secondary horseradish peroxidase-labeled antibody was
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added (0.2 mg mL-1 in PBS; 200 µL per well) and incubated for 1 h. After final washing, the
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substrate solution (200 µL per well) was added and the plates were shaken for about 15 min
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for color development. The substrate solution consisted of 25 mL substrate buffer (prepared 7 ACS Paragon Plus Environment
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with 46.0 mL potassium dihydrogen citrate and 0.1 g potassium sorbate in 1 L of water, pH
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3.8), 500 µL 3,3',5,5'-tetramethylbenzidine stock solution (375 mg in 30 mL of
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dimethylsulfoxide) and 100 µL 1% hydrogen peroxide. The enzyme reaction was stopped by
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adding 100 µL of 5% sulfuric acid per well. The absorbance was read at 450 nm by a
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microplate reader (Synergy HT, Bio-Tek Instruments).
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Data analysis
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A four-parameter logistic function (Eq. 1) was used for a regression analysis of the calibration
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curves:
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Y =
233
where [DCF] is the concentration of diclofenac, and Y is either the upconversion
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luminescence or the absorbance at 450 nm. Eq. 1 yields the maximum (Ymax) and background
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(Ybg) signal, the DCF concentration that reduces (Ymax-Ybg) by 50 % (IC50) and the slope at the
236
inflection point (s). All measurements were made at least in triplicate. The concentration of
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DCF in real water samples was determined by utilizing an inverse function of Eq. 1 and the
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limit of detection (LOD) was defined as before:26
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Y(LOD) = 0.85 × (Ymax - Ybg) + Ybg
Ymax − Ybg [ DCF ] 1 + IC 50
s
+ Ybg
(1)
(2)
240 241
Results and Discussion
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Surface modification and characterization of UCNPs
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The development of a competitive upconversion immunoassay (ULISA) for the detection of
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DCF (Figure 1) critically depends on the design of the luminescent reporter that replaces the
245
conventional enzyme amplification steps.30 Oleic acid-capped UCNPs were coated with a
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silica shell exposing carboxylic acid functional groups on the surface. The carboxyl groups
247
improve the dispersibility in water and serve as attachment sites for subsequent conjugation
248
steps. We previously described a one-step water-in-oil microemulsion protocol for coating the
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surface of small UCNPs (~12 nm in diameter) with a carboxylated silica shell that showed
250
only a weak upconversion luminescence.25 For the immunoassay, we have synthesized larger
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UCNPs of 42.5 ± 4.9 nm in diameter (Supporting Information Figure S1) that are much
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brighter because they are less affected by surface quenching effects.31 The one step silica8 ACS Paragon Plus Environment
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coating protocol, however, resulted in aggregation when directly applied to bigger
254
nanoparticles. Therefore, we developed a two-step protocol to prepare a thicker, compact and
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more stable silica shell on the surface of UCNPs.32 First, TEOS was added to the
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microemulsion to generate a thin layer of bare silica (2.4 ± 0.4 nm, Supporting Information
257
Figure S2). This step alone was not sufficient to prevent aggregation. Therefore, TEOS was
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added for a second time, which changed the thickness of the silica shell only slightly. The
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second carboxylation step ensured an excellent dispersibility of COOH-UCNP in water.33 The
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total diameter of COOH-UCNPs was consistent as determined by transmission electron
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microscopy (TEM: 46.9 ± 5.0 nm; Supporting Information Figure S3 and Figure 2A) and
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atomic force microscopy (AFM: 45.4 ± 7.6 nm; Supporting Information Figure S4). Dynamic
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light scattering measurements confirmed an increase of the hydrodynamic diameter from 55
264
nm to 65 nm after silica coating (Supporting Information Figure S5).
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The optimized COOH-UCNPs were then conjugated to a secondary anti-IgG antibody via
266
standard EDC/sulfo-NHS chemistry (Figure 2B).34,35 A low concentration of COOH-UCNP
267
was utilized to prevent that one antibody molecule binds to several UCNPs, which would lead
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to aggregation. The conjugates were characterized by agarose gel electrophoresis (Figure 2C),
269
dynamic light scattering (DLS), zeta potential measurements and FT-IR spectroscopy
270
(Supporting Information Figures S6-S8). The lowest degree of aggregation was observed
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when the concentration of COOH-UCNPs in the reaction mixture was 1 mg mL-1. The
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conjugation of the secondary antibody reduced the negative surface potential of the COOH-
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UCNPs as shown by zeta potential measurements and led to a stronger retardation in the
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agarose gel.36 The shift of the electrophoretic mobility was linearly dependent on the ratio of
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IgG molecules per UCNP and indicated the degree of surface modification (Figure 2D).
276
Additionally, larger aggregates of nanoparticles remained in the gel pockets and could not
277
enter the agarose matrix. Sample IgG-UCNP-33:1 shows a main fraction of monodisperse
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bioconjugates separated as a distinct band and a smaller fraction of slowly moving
279
components, which are probably partially aggregated and crosslinked bioconjugates. This
280
result is consistent with a bimodal particle distribution and a higher polydispersity index
281
observed in the DLS measurement.
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The IgG-UCNP conjugates were purified from an excess of unbound secondary anti-mouse
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IgG and components of the reaction mixture by differential centrifugation. At first, the
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bioconjugates were centrifuged at 10,000 g, which, however, led to strong nanoparticle
285
aggregation (Figure 2C). When the centrifugal speed was reduced to 4,000 g followed by 9 ACS Paragon Plus Environment
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short sonication, purified and monodisperse IgG-UCNP were obtained. Further lowering of
287
the centrifugal field was not efficient since the sedimentation of IgG-UCNP was too slow.
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The small retardation coefficient of UCNPs prepared with the lowest amount of IgG (IgG-
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UCNP-3:1, Figure 2C, lane V) indicated an insufficient surface modification. Therefore, this
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bioconjugate was not used for the following ULISA experiments.
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292 293 294 295 296 297 298 299 300 301
Figure 2. Preparation and characterization of IgG-UCNP conjugates. (A) TEM image of silica-coated UCNPs exposing carboxyl groups on the surface (COOH-UCNPs, Supporting Information Figure S3). (B) The carboxyl groups are activated by EDC/sulfo-NHS and conjugated to anti-mouse IgG. (C) The conjugates are prepared by using different ratios of anti-mouse IgG and COOH-UCNPs (I/II: 33 to 1; III/IV: 7 to 1; V/VI: 3 to 1, VII/VIII: no IgG). Each sample is centrifuged either with 4,000 g (I, III, V, VII) or with 10,000 g (II, IV, VI, VIII) and characterized by agarose gel electrophoresis. The migration distance (∆) is indicated with red lines. (D) The relative electrophoretic mobility (∆ratio[x]/∆no IgG) of the conjugates is linearly dependent on the ratio of IgG molecules per UCNP.
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Design of Upconversion-linked immunosorbent assay (ULISA)
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In a competitive immunoassay, a low concentration of coating antigen ensures that the free
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analyte can compete efficiently for the binding sites of the detection antibodies. On the other
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hand, the signal generation has to be strong enough for a reliable readout. Here, we prepared
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two different coating conjugates consisting of BSA-DCF. The conjugates were analyzed by
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MALDI-TOF mass spectrometry, which showed a coupling density of either 5.7 or 10 DCF
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residues per BSA molecule (Supporting Information Figures S9-S11). When the conjugate 10 ACS Paragon Plus Environment
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with the higher degree of derivatization was used for coating in the immunoassay, the signals
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were – as expected – about twice as high but also showed stronger signal fluctuation and a
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hook effect (Figure 3A), which may be the consequence of two binding sites of IgG
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molecules forming cyclic complexes (Supporting Information Figure S12).37 By contrast, the
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conjugate exposing 5.7 DCF residues per BSA molecule yielded more stable signals and a
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slightly lower IC50 value (1.2 ng mL-1 compared to 1.5 ng mL-1) and a lower detection limit
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for DCF. Consequently, this coating conjugate was used in all further experiments. An
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optimal signal generation was observed with a coating concentration of 1 µg mL-1
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(Supporting Information Figure S13).
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Figure 3. ULISA optimization. (A) Microtiter plates are coated with 1 µg mL-1 of BSA carrying either 10 (□) or 5.7 (○) DCF residues. (B) The upconversion luminescent (UCL) signal is generated by using 10 µg mL-1 of IgGUCNP-33:1 (○) or IgG-UCNP-7:1 (□), respectively. (C) The detection of DCF is optimized by using the monoclonal anti-DCF antibody in concentrations of 0.5 µg mL-1 (○) (IC50: 0.68 ng mL-1), 0.25 µg mL-1 (□) (IC50: 0.23 ng mL-1), 0.1 µg mL-1 (∆) (IC50: 0.13 ng mL-1) or 0.02 (◊) µg mL-1 (IC50: 0.08 ng mL-1). Error bars represent standard deviations in upconversion signals from three replicate wells.
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The competition step including free DCF and anti-DCF detection antibody was performed
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in analogy to a sensitive conventional ELISA.26 Only the enzyme-mediated color generation
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was replaced by an IgG-UCNP conjugate as a direct luminescent reporter (Figure 3B). The
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higher degree of UCNP surface coverage (IgG-UCNP ratio of 33:1 compared to 7:1)
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increased the maximum signal intensity by a factor of five although both conjugates were
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prepared with a molar excess of IgG molecules per nanoparticle. This difference can be
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explained because not every surface-conjugated antibody may have the right orientation or be
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fully functional in order to bind efficiently to the primary antibody. Consequently, a higher
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degree of derivatization resulted in a proportionally higher number of functional antibodies.
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On the downside, using IgG-UCNP-33:1 resulted in strong signal fluctuations as well as a
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hook effect, which impedes the reproducible determination of DCF. It should also be noted
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that the degree of surface substitution did not significantly affect IC50 or the LOD, and a
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concentration of 10 µg mL-1 IgG-UCNP-7:1 resulted in the most reproducible upconversion
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signal generation (Supporting Information Figure S14)
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In contrast to the UCNP-bound secondary antibody, the primary anti-DCF antibody is
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directly involved in the competition step. Figure 3C shows that both the upconversion signal
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intensity and the IC50/LOD for DCF strongly depend on the antibody concentration. A higher
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primary antibody concentration leads to a higher signal intensity because more antibodies can
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bind to the DCF-BSA coating conjugate, but they also consume a larger amount of free DCF
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and thus deteriorate the assay sensitivity. A concentration of 0.25 µg mL-1 primary anti-DCF
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antibody yielded an optimal balance between signal generation and sensitivity for the
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determination of DCF and was used in all further experiments.
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Calibration and sensitivity of ULISA and ELISA
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For each type of competitive immunoassay it is necessary to find the optimal balance between
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detection sensitivity for an analyte and signal development. It should also be noted that a high
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affinity and a low cross-reactivity of the primary antibody are the most distinctive features
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that determine the sensitivity and specificity of the analyte detection. Figure 4 shows
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calibration curves of ULISA and ELISA recorded under similar conditions and using the same
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anti-DCF primary antibody. In both cases a signal to background ratio (Ymax/Ybg) of 5:1 was
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adjusted to achieve the most sensitive detection of DCF but also to obtain a reliable signal
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generation. The competitive ULISA (LOD: 0.05 ng mL−1 / 170 pM) has a five times higher
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detection limit than a conventional ELISA (LOD: 0.01 ng mL−1/ 34 pM) but allows for an
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easier and faster signal generation. As the detection sensitivity is ultimately dependent on the
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anti-DCF antibody, it can be expected that the ULISA can be further optimized by developing
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brighter UCNPs reporter conjugates.
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Competitive immunoassays for small molecules are typically less sensitive than sandwich
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immunoassays where the signal generation is directly proportional to the analyte
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concentration. The highest sensitivity was described for the detection of Schistosoma
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circulating anodic antigen by using micron-sized upconversion particles in a lateral flow assay
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(LOD: 0.01 ng mL-1 / 0.1 pM).13 This particular analyte displays repetitive surface epitopes
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and facilitates binding of several primary antibodies per analyte molecule. The competitive
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immunoassay for DCF affords a similar sensitivity as a magnetic bead-based competitive
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immunoassay for the detection of aflatoxin that was reported to reach an LOD of 0.01 ng mL-1
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(50 pM) under optimal conditions.16 The additional magnetic separation step, however,
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demands a more sophisticated instrumentation and is more time consuming. 12 ACS Paragon Plus Environment
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Figure 4. Normalized calibration curves of ULISA (□, replotted red curve from Figure 3C; IC50: 0.23 ng mL−1, LOD: 0.05 ng mL−1) and ELISA (○, IC50: 0.05 ng mL−1, LOD: 0.01 ng mL−1). Error bars represent standard deviations of three replicate wells.
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Detection of diclofenac in real water samples
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Two surface water samples and drinking water were collected in Southern Bavaria and the
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matrix was analyzed (Supporting Information Table S1) to assess possible interferences with
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the detection of DCF. These interferences should be as low as possible because matrix effects
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can suppress the immunoassay signal and lead to an overestimation of analyte concentrations.
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The monoclonal primary antibody 12G5 is resistant to matrix interferences over a wide pH
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range, humic acid concentrations up 20 mg L-1 and Ca2+ concentrations up to 75 mg L-1 as
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described earlier.26 The drinking water sample from Munich, however, contained relatively
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high Ca2+ and Mg2+ concentrations of 110 mg L-1 in total, which is probably the reason for a
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signal suppression (defined as 100 × (Ysample - Ybg) / (Ymax - Ybg)) to 60±7 % in the ULISA and
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73±10 % in the ELISA in the undiluted samples without DCF. By contrast, the surface water
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samples contained less Ca2+ and Mg2+ and were less affected by signal suppression.
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The concentration of DCF was too low to be detectable in the unspiked water samples.
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Thus, each sample was additionally spiked with either 1 ng mL-1 or 10 ng mL-1 of DCF. The
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spiked samples were typically diluted at least by a factor of three prior to the immunoassay to
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keep matrix effects to a minimum. Table 1 shows the concentrations of DCF as determined by
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ULISA, ELISA and LC-MS. The ULISA led to slightly stronger deviations from the spiking
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concentration compared to the ELISA because the matrix may also have an impact on the
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binding of the nanoparticulate luminescent reporter unit, which is relatively large compared to
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the enzyme antibody conjugate used for the ELISA. These differences in the immunoassay
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performance are subject to further investigation and will be optimized to unfold the full
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potential of the ULISA for the background-free detection of analytes.
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Table 1. Detection of DCF in unspiked and spiked real water samples. Sample
Lake Wörthsee
River Würm
Munich tap water
Spiked (ng mL-1)
ULISA (ng mL-1)
ELISA (ng mL-1)
LC-MS (ng mL-1)
-
