Switchable lanthanide luminescent binary probes in efficient single nucleotide mismatch discrimination

Sensors and Actuators B 211 (2015) 297–302

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Switchable lanthanide luminescent binary probes in efficient single nucleotide mismatch discrimination Ulla Karhunen a,∗ , Eeva Malmi a , Ernesto Brunet b , Juan Carlos Rodríguez-Ubis b , Tero Soukka a a b

Department of Biotechnology, University of Turku, Tykistökatu 6 A 6th Floor, 20520 Turku, Finland Department of Organic Chemistry, Autonomous University of Madrid, 28079 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 26 October 2014 Received in revised form 9 January 2015 Accepted 24 January 2015 Available online 31 January 2015 Keywords: Time-resolved fluorescence Single nucleotide polymorphism Switchable luminescence Chelate complementation

a b s t r a c t Switchable lanthanide luminescence is a novel proximity-based binary probe technology wherein two oligonucleotide probes are labelled either with lanthanide ion carrier chelate or light-absorbing antenna ligand. A highly luminescent complex is formed when the two label moieties are brought in close contact by adjacent hybridization of the labelled probes to a target sequence. Herein we report how hybridization distance between the labelled probes and single nucleotide mismatches in the target sequence affect to the luminescence intensity of the complex formed from the label moieties. The highest luminescence intensity was observed when the probes were hybridized to full matched target at a distance of 4–10 nucleotides, a fact that indicates the lanthanide complex requires a certain space to be formed. At optimal hybridization distance between the labelled probes the maximal discrimination efficiency between a full matched and a single mismatched target was 1400-fold. Furthermore, computer-based modelling of the luminescent complex formation was in good agreement with the experimental results. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Single nucleotide polymorphisms (SNPs) represent the most common class of variations in the human genome [1]. For genotyping of single mutations, e.g. restriction fragment length polymorphism, differential hybridization, melting temperaturebased separation, allele specific amplification and DNA sequencing have been used [2]. In high-throughput analysis of SNPs required in genetic research, DNA microarrays are commonly used [3]. High specificity is also needed when variants of highly conserved genes like 16SrRNA are used for separation of a target from a non-target micro-organism [4]. Differential hybridization utilizes two oligonucleotide probes that are complementary to the mutated and wild-type alleles. While designing an oligonucleotide probe for differential hybridization assay, the length of the probe is an important parameter that determines the selectivity of the hybridization [5]. The longer the probe, the higher its binding affinity and thus the lower its specificity. The mismatch discrimination can be improved by locked nucleic acids [6,7], chaotropic agents [8], low salt concentration and measurement at elevated temperatures.

∗ Corresponding author. Tel.: +358 2 333 8042. E-mail address: ulla.karhunen@utu.fi (U. Karhunen). http://dx.doi.org/10.1016/j.snb.2015.01.092 0925-4005/© 2015 Elsevier B.V. All rights reserved.

Another approach is the use of binary probes [9]. In binary probe system, the selectivity is enhanced by the requirement of two simultaneous hybridization events which results in a detectable signal. Binary probe systems using Förster resonance energy transfer [10] (FRET) from a donor to an acceptor using short-lived fluorophores such as cyanine dyes and fluorescein have been used in SNP diagnostics [11–13]. However FRET has some limitations: (i) excess fluorescence background from autofluorescence, (ii) radiative energy transfer to the acceptor and (iii) direct excitation of the acceptor and donor emission cross-talk at the acceptor emission measurement wavelength, that may cause elevated background and thus compromise assay sensitivity. Luminescence from lanthanide ions such as Eu3+ and Tb3+ has unique properties like long emission lifetime, sharp emission peaks and large Stokes’ shift. However, direct excitation of a lanthanide ion is quite inefficient and therefore the ion is typically chelated to a light harvesting ligand that absorbs and transfers the excitation energy to the lanthanide ion and protects the ion from the quenching effects of water molecules. The unique properties of lanthanide luminescence have enabled the development of sensitive detection methods for nucleic acid testing [14] and immunoassays [15]. In switchable lanthanide luminescence technology two nonluminescent label moieties, a light absorbing antenna ligand and a lanthanide ion carrier chelate, can self-assemble to form a highly luminescent mixed-chelate complex when they are brought

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Fig. 1. Schematic representation of the switchable lanthanide luminescence assay principle and sequences of the model oligonucleotide probes. Labelled oligonucleotide probes are non-luminescent in the absence of complementary target oligonucleotide. After hybridization of a labelled probe pair adjacently on the target oligonucleotide, the label moieties form a luminescent complex which can be excited at 340 nm and the long lifetime emission can be measured in time-resolved mode at 615 nm. Amino modifications for label moiety coupling: ¤: Amino C3; (T#): Amino C2dT; (T*): Amino C6dT; *: Amino C12.

together in close proximity. Herein, the luminescent complex is formed after adjacent hybridization of a 3 -ion carrier chelate conjugated probe and a 5 -light absorbing antenna ligand conjugated probe on the complementary target nucleic acid (Fig. 1). Here we have studied in detail how the distance between the two labelled oligonucleotides of the binary probe system and single nucleotide mismatches in the target sequence affect the luminescence intensity of the mixed lanthanide chelate complex. Furthermore, computer-based modelling of the luminescent complex formation was performed and the results were compared to the experimental luminescence intensities. 2. Materials and methods 2.1. Materials The oligonucleotide probes presented in Fig. 1 were purchased from Sigma–Aldrich (St. Louis, MO) and oligonucleotide targets presented in Table 1 from Biomers.net GmbH (Ulm, Germany). Upon arrival the targets were dissolved in storage buffer (10 mM Tris, pH 7.5, 50 mM NaCl and 10 ␮M EDTA) and the DNA concentrations were confirmed by absorbance at 260 nm. The assay buffer used in distance measurement consisted of 50 mM Tris, pH 7.6, 300 mM NaCl, 0.05% Tween-20, 0.05% NaN3 and 50 ␮M DTPA. The buffer used in the single nucleotide mismatch measurements was similar except the NaCl concentration was 50 mM. Hybridization reactions were performed on low fluorescence 96-well Maxisorp microtitration plates purchased from Nunc (Roskilde, Denmark) unless otherwise mentioned. 2.2. Oligonucleotide probes The model probe and target sequences used in this study were designed to obtain hybridization to the full matched targets already at room temperature. However, in order to enhance the selectivity of the probe pair towards detection of single nucleotide mismatches, the length of probes A1 and B1 was decided to be

only 11 nucleotides and the mismatches on the target oligonucleotides were positioned at the hybridization site of these two probes. The amino linker lengths and positions chosen for label moiety coupling on the two probe pairs showed highest (probe pair A) and lowest (probe pair B) luminescence intensities in preliminary experiments among eighteen linker length and position combinations (data not shown). The selected probe pair A contained a primary amine separated by aliphatic three-carbon chain (amino C3) from the 3 phosphate for ion carrier chelate conjugation and an internal amino C2 on penultimate thymine at 5 for antenna ligand conjugation (Figs. 1 and A1). The amino linker positions were vice versa on the probe pair B, where the ion carrier chelate was conjugated to internal amino C6 on ultimate thymine at 3 and antenna ligand to amino C12 at 5 phosphate. The two 3 -aminomodified probes (A1 and B1 in Fig. 1) were labelled with isothiocyanate-activated form of the ion carrier chelate DOTA-EuIII [2,2 ,2 -(10-(3-isothiocyanatobenzyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)tri(acetate)europium(III)] [16] and the two 5 -aminomodified probes (A2 and B2 in Fig. 1) were labelled with isothiocyanate-activated form of the light absorbing antenna ligand [4-((isothiocyanatophenyl)ethynyl)pyridine-2,6dicarboxylicacid] [17] as described previously [18].

2.3. Effect of probe hybridization distance on luminescence intensity The two labelled probe pairs (A and B; 20 nM) and full matched targets with 0–30 thymines between the probe hybridization positions (FM0–FM30; 10 nM) were combined in the assay buffer (total volume of 60 ␮L) as three replicas. The hybridization reactions were first incubated at +50 ◦ C for 30 min and then at room temperature in slow shaking for 60 min before luminescence measurement in time-resolved mode with an EnVision Multilabel Plate Reader (PerkinElmer Life and Analytical Sciences, Waltham, USA). The lanthanide complex was laser-excited at 337 nm and the emission at 615 nm was measured with 400 ␮s delay and 400 ␮s measurement window.

Table 1 Full matched (FM) and single nucleotide mismatched (MM) target sequences. Oligonucleotidea,b

Sequence 5 → 3 a,b,c

Nc

FMn MMm MMm MMm MMm MMm

GCA AGA ATG GCG CAA (T)n ACG ATT GTA AG GCA AGA ATG GCG CAA (T)m NCG ATT GTA AG GCA AGA ATG GCG CAA (T)m ANG ATT GTA AG GCA AGA ATG GCG CAA (T)m ACN ATT GTA AG GCA AGA ATG GCG CAA (T)m ACG NTT GTA AG GCA AGA ATG GCG CAA (T)m ACG ANT GTA AG

T, C, or G A, T, or G A, T, or C T, C, or G A, C, or G

a b c

1N 2N 3N 4N 5N

n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30. m = 1 or 6. N denotes a single nucleotide mismatch base at the target sequence.

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2.4. Single nucleotide mismatch discrimination Full matched target oligonucleotides with one and six thymines between the probe hybridization positions (FM1 and FM6; 10 nM) and corresponding single nucleotide mismatched targets (MM1 1N–MM1 5N and MM6 1N–MM6 5N; 10 nM) were combined with the labelled probe pairs (A and B; 20 nM) in the mismatch assay buffer (total volume of 60 ␮L) as three replicas. The incubation and luminescence measurement were performed like described in Section 2.3. 2.5. Effect of temperature on luminescence intensity For melting temperature measurement a full matched target (FM6) and six corresponding mismatched targets (MM6 1N and MM6 4N) were combined with the labelled probe pair A. The target oligonucleotides were 50 nM and the probe pair 55 nM in the mismatch assay buffer (total volume of 120 ␮L). The hybridization reactions were incubated in microcentrifuge tubes at +50 ◦ C for 30 min and then at room temperature for 1 h before measurement in a quartz cuvette from Hellma GmbH (Müllheim, Germany) with a Varian Cary Eclipse fluorescence spectrophotometer equipped with a thermostated single-cuvette holder (Varian Scientific Instruments, Mulgrave, Australia). The luminescence was measured in one degree intervals from 20 ◦ C to 55 ◦ C with average temperature ramp rate of 1.0 ◦ C/min using 340 nm excitation with 20 nm slit, 615 nm emission with 10 nm slit, 100 ␮s delay and 700 ␮s gate. A sigmoidal fit to Boltzmann equation was used to obtain melting temperatures of the luminescent complexes formed of the probe pair with the full matched and mismatched targets using Origin8 programme from OriginLab (Northampton, MA, USA). 2.6. Molecular modelling The model building of the different structures and the corresponding calculations was performed by means of the HyperChem programme (HC) (http://www.hyper.com) in Windows-based PC computers. All energy minimizations were performed using the molecular mechanics AMBER force field [19] with default parameters and the Polak-Ribiere algorithm with 0.01 RMS value for convergence. The hybridization systems A and B (Figs. 1 and A1) were assembled by means of HC’s nucleotide database and the nucleic acid builder using default parameters (B helix and 2 -endo sugar forms). The structure of the 1,4,7,10-tetraazacyclododecane1,4,7-triacetate (DOTA) derivative was built from X ray literature data of similar compounds [20] and was left unchanged throughout the calculations using the necessary restraints. The label moieties were attached at the right positions on the probes (Figs. 1 and A1) and were given a preliminary minimization round. The complexing ends were then gradually brought in together by means of the suitable distance and angle restrictions. The Eu and the three chelating atoms from the pyridine dicarboxylates were restricted to a plane. In the simulated interaction between the pyridine dicarboxylate of probes A2 and B2 and the DOTA-chelated lanthanide metal, ˚ the final Eu–N and Eu–O bonds were constrained to 3.0 and 2.5 A, respectively, with a force constant of 1.5. After energy minimization the Eu–N and Eu–O bonds ended up with average distances ˚ The final structures and energy values were obtained of ca. 3.5 A. limiting the energy minimization to the thymines (those carrying the amino linker or next to the amino linker modified terminal phosphate) onto which the label moieties were conjugated, the latter themselves and the polythymine gap. The fragment was then freely allowed to be minimized by the HC programme keeping the aforementioned chelating restrictions. The final energy values of the fragment were divided by the corresponding number of atoms giving rise to an average energy-per-atom value that was used as

Fig. 2. Luminescence intensity of the lanthanide complexes formed from labelled probe pairs (black square: pair A; white square: pair B) hybridized to full matched target sequences (FM0–FM30) at increasing distance between the probes. The luminescence intensities were obtained by subtracting the background luminescence with zero concentration of target oligonucleotide from the raw signals with different target oligonucleotides. Error bars indicate the standard deviation of three replicas.

a follow-up of the fragment strain and a qualitative indication of complex stability. 3. Results and discussion 3.1. Effect of probe hybridization distance on luminescence intensity In the probe hybridization distance experiment the labelled probes A1 and A2 formed a probe pair with short three- and two-carbon (C3 and C2) linkers, respectively, between the oligonucleotides and the label moieties whereas labelled probes B1 and B2 formed a probe pair with long linkers (C6 and C12, respectively). The luminescence intensity resulting from labelled probe pairs with increasing hybridization distance is presented in Fig. 2. Interestingly, the pair B results in low luminescence intensity when the probes are hybridized adjacently or at a short distance to each other. When the distance between labelled probes increases, the luminescence intensity increases also. On the contrary, the pair A has relatively high luminescence intensity even at short hybridization distances but similarly to the pair B, the highest intensities being achieved when the probe hybridization sites are separated by approximately 4–10 thymines. 3.2. Single nucleotide mismatch discrimination In order to study the effect of single nucleotide mismatches in the target sequences on the luminescence intensity of the lanthanide complex, mutations were placed at five positions on the FM1 and FM6 target oligonucleotides complementary to probes A1 and B1. The luminescence intensities are presented in Fig. 3a and c. With FM1 full match and corresponding mutated targets, the probe pair A has higher luminescence intensities when compared to the intensities obtained from the probe pair B. Six thymines separating the probe hybridization positions results in slightly higher mismatch discrimination efficiency than one separating thymine (Fig. 3b and d). The maximum discrimination efficiencies obtained in this work (1400-fold intensity of the full match as compared to

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Fig. 3. Luminescence intensities (a and c) and signal with the full match target compared to the signal with mutated target (b and d) of the labelled probe pair A (black columns) and labelled probe pair B (grey columns). Bar charts (a) and (b): single thymine in the target separates the labelled probe pairs; (c) and (d): six thymines separate the labelled probe pairs. The error bars indicate standard deviation of three replicas.

the mismatched target) are outstanding when compared to the 7fold obtained by Ihara et al. [21] using a similar lanthanide complex and a probe pair and to the 10-fold by Kolpashchikov [22] using a molecular beacon combined with a binary probe pair. Mutations in the middle of the probe A1 and B1 hybridization site were discriminated more efficiently from the full matched targets whereas mismatches at positions 1 and 2 resulted in luminescence intensities closer to the full matched targets. This is in accordance with earlier studies of single nucleotide mutation discrimination accuracy [23–25]. 3.3. Effect of temperature on luminescence intensity Hybridization of probe pair A to FM6 and six single nucleotide mismatched targets (MM6 1T, 1C, 1G and MM6 4T, 4C, 4G) were analyzed in more detail. Luminescence intensity was measured as a function of temperature and a sigmoidal fitting was used to obtain the melting temperature of the luminescent complex (Table 2). For the MM6 4T and MM6 4C a Tm could not be calculated because their luminescence intensities were low and did not show a sigmoidal response in the measured temperature range. This confirms that the probe A1 only weakly hybridized to these mutated targets. Guanine mismatches with thymine, adenine and guanine are known for their enhanced stability [26,27] which is seen here as well, i.e. MM6 1G and MM6 4G displayed higher Tm s when compared to MM6 1T and C, and to MM6 4T and C, respectively. A similar phenomenon is seen in Fig. 3c where these guanine mismatched targets

also have higher luminescence intensities than the corresponding T and C mismatches. 3.4. Molecular modelling The modelling calculations assumed the polythymine gaps in the targets to hold the typical helix conformation equivalent to the double stranded region. Fig. A2 shows six example models of probe pairs A and B (Fig. A1) hybridized with full matched targets. In that arrangement the distance between the highlighted thymines (those carrying the amino linker or next to the amino linker modified terminal phosphate) increases from ca. 0.5 to 6 nm throughout

Table 2 Measured melting temperatures (Tm ) of the luminescent lanthanide complexes formed from the labelled probe pair A with full matched FM6 and six single nucleotide mismatched targets. Target oligonucleotide

Tm (◦ C)a

FM6 MM6 MM6 MM6 MM6 MM6 MM6

30.3 ± 0.06 26.3 ± 0.07 25.5 ± 0.13 28.3 ± 0.05 n/a n/a 23.8 ± 0.57

1T 1C 1G 4T 4C 4G

n/a, not applicable. a Errors show the standard deviation.

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Fig. 4. Models of the probe pairs A and B hybridized with targets FM5 and FM10 containing the indicated label moieties (highlighted in overlapping-spheres mode) in the appropriate geometry to form the expected luminescent complexes.

the series. Fig. A3 contains calculated models of the expected luminescent complexes at stable conformers when the linker ends are kept at maximal distance from each other. In the labelled probe pairs A and B the distances between linker ends were 3.1 and 4.7 nm (Fig. A3), respectively, a separation that roughly corresponds to those found in the hybridizations of the probe pairs with full matched target FM10 (Fig. A2). Fig. 4 shows the calculated models of the probe pairs A and B hybridized with full matched targets FM5 and FM10. In case of both the probe pairs and targets the luminescent complexes are formed with reasonable conformations. Both the labelled probe pairs A and B showed relatively high luminescence value (Fig. 2) when hybridized with FM15 despite the huge distances resulting between the thymines carrying the amino linker or thymines next to the amino linker modified terminal phosphate onto which the label moieties were conjugated (Fig. A2). This suggests that the single stranded polythymine gap is quite flexible allowing the complex formation in case of both the probe pairs. On the other hand, the lower luminescence with FM15 as compared to the shorter counterparts FM5 and FM10 can reasonably be understood based on entropic grounds since the larger the polythymine gap the more degrees of freedom must be overcome to achieve the luminescent complex. Interestingly, the largest differences in luminescence behaviour are observed for the probe pairs hybridized with the shortest targets (Fig. 2). The probe pair A displayed relatively high luminescence intensities, but its counterpart B showed negligible intensities in the case of hybridization with targets FM0, FM1 and FM2. Fig. A1 and the models in Fig. 4 illustrate that the probes B1 and B2 share long alkyl chains whose zig-zag conformations make it difficult for the pyridine dicarboxylate (probe B2) and DOTA-Eu (probe B1) to reach the proper luminescent complex arrangement. Table 3 summarizes the calculations performed on the probe hybridizations over their pertinent fragments containing the label moieties, the thymines carrying the amino linker or thymines next to the amino linker modified terminal phosphate onto which the label moieties were conjugated, and the polythymine gap. Calculations do qualitatively agree with the observed luminescence intensity since the average energy per atom for hybridization with targets FM0, FM1

Table 3 Calculated energy per atom (kcal mol−1 ) of the minimized fragment containing the label moieties, the thymines carrying the amino linker or thymines next to the amino linker modified terminal phosphate onto which the label moieties were conjugated, and the polythymine gap for the probe pairs A and B hybridized with six FM targets. Target

FM0 FM1 FM2 FM5 FM10 FM15

Energy (kcal mol−1 )

and FM2 are higher (less stable) for the probe pair B, the case showing negligible luminescence (Fig. 2). The lowest energy-peratom values found for hybridization of FM5 and FM10 with probe pair A are in relatively good agreement with the luminescence intensities observed (Fig. 2). On the other hand, the steady decrease of calculated energy-per-atom throughout the probe pair B series fails to agree with the experimental results. One should be aware that the performed, simple calculations take only into account the internal strain energy (see Section 2) of a single molecule in the gas phase and other important factors like entropic contributions or, perhaps more importantly, differential salvation (the luminescence deleterious effect of water molecules in the coordination sphere of the emitting metal is well known [28]) are not considered.

4. Conclusions We have previously used switchable lanthanide luminescence for homogeneous nucleic acid [17,29,30] and protein [16,31] detection in solution and in homogeneous DNA-based array format [18]. Luminescent complex has previously been reported to form from adjacent hybridization of binary probes on a target nucleic acid in solution [32–34]. Self-assembly of a luminescent lanthanide complex has also been presented resulting from the enforced close proximity of a sensitizer and europium(III) complex immobilized on a glass [35] and gold [36] surfaces. This study shows that switchable lanthanide luminescence is suitable also for single nucleotide mutation detection. The luminescence intensity of a probe pair labelled with switchable lanthanide luminescence label moieties can be increased by positioning the labelled probe pair at a distance of 4–10 nucleotides which indicates that the formation of the lanthanide complex requires certain space between probe hybridization positions. The maximum discrimination of 1400-fold between a full matched and a single mutated target presented here is outstanding among fluorescence-based methods. The genotyping of clinical samples is typically performed after DNA extraction and nucleic acid amplification. The presented homogeneous method could be utilized for example in the analysis of such amplification products. A weak co-operative effect between the two labelled probes and the binding of the label moieties to each other may stabilize the correct hybridization and partly explain the excellent discrimination. Computer-based modelling of the formation of the luminescent lanthanide complex was in good agreement with the experimental results.

Acknowledgements

Probe pair A

Probe pair B

1.04 1.09 0.99 0.89 0.81 1.01

1.25 1.16 1.09 0.99 0.85 0.73

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under Grant Agreement no. 259848 and the Academy of Finland, Grant number 132007. Financial support from ERCROS S.A. (Aranjuez, Spain) through the UAM-ERCROS Chair for Pharmaceutical Chemistry is gratefully acknowledged.

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Biographies Dr. Ulla Karhunen received her PhD in molecular biotechnology and diagnostics from University of Turku in 2014 where she worked with switchable lanthanide luminescence and sensitive assays for nucleic acids and other biomolecules. She currently works as a coordinator at the Turku Centre for Biotechnology. Eeva Malmi currently works as a research chemist at Kaivogen Oy, a biotechnology company located in Turku, Finland. Her work is related on a novel label technology based on photon upconversion (www.upcon.fi). Alongside the work she is completing her M.Sc. on Molecular Biotechnology and Diagnostics at the University of Turku. The focus of her thesis is on the use of switchable lanthanide luminescence technology for homogeneous protein detection. Dr. Ernesto Brunet-Romero is currently full Professor of Organic Chemistry at Autonomous University of Madrid (UAM) since 1986. He completed his PhD at UAM with honours in 1983. Fulbright and NATO fellow (University of North Carolina at Chapel Hill with Prof. Eliel) in 1984–1985. He has worked in several stereochemical problems of organic molecules and more recently in the development of organic–inorganic materials based on zirconium phosphate where the organic moieties displayed unusual properties under confinement. He has been also involved in the development of lanthanide chelates for luminescence signalling. He is director of the research group LUMILA and of the sponsored Chair UAM-Ercros in Pharmaceutical Chemistry. Professor Juan Carlos Rodriguez-Ubis received his PhD from UAM, where he worked on the synthesis of natural products. In 1983, he moved to France as a postdoctoral fellow with Prof. Lehn (Universite Louis Pasteur, Strasbourg) doing research on synthetic cryptands based on pyridine subunits as light antennae for lanthanide emission. In 1986, he became Organic Chemistry Professor at the Universidad Autonoma de Madrid where he started his independent research career in 1992. His current research interest involves supramolecular processes associated with photoactive lanthanide complexes and layered organo-inorganic materials based on zirconium phosphate. Professor Tero Soukka is currently a Professor of Biotechnology (In vitro Diagnostics) at University of Turku. He obtained a Ph.D. in 2003 from University of Turku and worked thereafter first as post-doctoral researcher and from 2007 to 2009 as Academy of Finland research fellow establishing a research group on bioanalytical assay technology research. His current research interests are in development and applications of long-lifetime lanthanide luminescence and lanthanide-based photon upconversion for point-of-care immunoassays and multiplexed nucleic acid amplification tests.