Oligonucleotide Array-in-Well Platform for Detection and Genotyping

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Oligonucleotide Array-in-Well Platform for Detection and Genotyping Human Adenoviruses by Utilizing Upconverting Phosphor Label Technology Minna Ylih€arsil€a,*,†,‡ Timo Valta,† Maija Karp,† Liisa Hattara,§ Emilia Harju,^ Jorma H€ols€a,^,z,|| Petri Saviranta,§ Matti Waris,‡ and Tero Soukka† †

Department of Biotechnology, University of Turku, Tykist€okatu 6A, FI-20520 Turku, Finland, Department of Virology, University of Turku, Kiinamyllynkatu 13, FI-20520 Turku, Finland, § Medical Biotechnology Centre, VTT Technical Research Centre of Finland, It€ainen Pitk€akatu 4C, FI-20520 Turku, Finland, ^ Department of Chemistry, University of Turku, FI-20014 Turku, Finland, z Turku University Centre for Materials and Surfaces (MatSurf), Turku, Finland, Departamento de Química Fundamental, Instituto de Química, Universidade de S~ao Paulo, Av. Prof. Lineu Prestes, 748, CEP05508900, S~ao Paulo-SP, Brazil

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‡

ABSTRACT: We have developed a robust array-in-well test platform based on an oligonucleotide array, combining advantages of simple instrumentation and new upconverting phosphor reporter technology. Upconverting inorganic lanthanide phosphors have a unique property of photoluminescence emission at visible wavelengths under near-infrared excitation. No autofluorescence is produced from the sample or support material, enabling a highly sensitive assay. In this study, the assay is performed in standard 96-well microtiter plates, making the technique easily adaptable to high-throughput analysis. The oligonucleotide array-in-well assay is employed to detect a selection of ten common adenovirus genotypes causing human infections. The study provides a demonstration of the advantages and potential of the upconverting phosphor-based reporter technology in multianalyte assays and anti-Stokes photoluminescence detection with an anti-Stokes photoluminescence imaging device.

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he interest in developing new diagnostic methods based on arrays of multiple probes to detect and subtype a wide range of different infectious agents is increasing. Microarray techniques are flexible and have high throughput capability. Therefore, they hold great potential for pathogen detection, identification, and genotyping in molecular diagnostic laboratories.1,2 Human adenoviruses (hAdV) are pathogens causing a broad spectrum of different diseases that affect respiratory, ocular, and gastrointestinal systems. In humans, there are currently 52 described adenovirus serotypes categorized into six subgenera (A-F) based on biological and molecular criteria.3,4 A hybridizationbased microarray will permit multiplexed, rapid, and accurate detection of adenoviruses and their genotypes.5,6 There are many fluorescent dyes available for direct and indirect labeling of target DNA via enzymatic or chemical modification. A fluorescent dye can be coupled into primers or nucleoside triphosphates and then enzymatically incorporated to DNA using PCR amplification.7 It is also possible to introduce specific functional groups or ligands into synthesized oligonucleotides, which provide targets for subsequent chemical fluorescent labeling. Organic fluorescent dyes and their combinations are typically used for the detection of hybridized targets on microarrays; e.g., Cy3 and Cy5 are two widely utilized dyes for genome-based array experiments.6,8 However, organic fluorophores r 2011 American Chemical Society

suffer from dye photobleaching, low signal intensities, spectral overlaps, and poor photostability.9 The photostability of the fluorescent labels is important because dye photobleaching can reduce significantly the available time for imaging. Therefore, it is highly desirable to develop new detection technologies utilizing photostable fluorescent probes for microarray-based assays. Different kinds of novel nanostructures, such as fluorophoredoped nanoparticles,10 semiconductor quantum dots,11 and inorganic and organic nanoparticles,12,13 have gained a lot of attention in recent years in microarray technology. In this study, we have used inorganic lanthanide-doped upconverting phosphors (UCPs) as novel reporters. These phosphors are submicrometer-sized particles consisting of a crystalline host doped with selected rare earth ions. UCPs have a unique feature of being capable to convert low-energy infrared radiation to high-energy visible light via absorption of multiple photons.14 Photon upconversion is exceptional in that no fluorescence background is produced from any biological material at the visible wavelengths by the infrared excitation and the anti-Stokes photoluminescence can thus be measured entirely Received: December 1, 2010 Accepted: January 6, 2011 Published: January 24, 2011 1456

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Analytical Chemistry free of autofluorescence and scattered infrared radiation, enabling a highly sensitive assay.14-18 The optical properties of the UCPs are unaffected by their environment because upconversion occurs within the host crystal. The only instrument sources of background signals are the stray light and detector noise. In addition, UCPs are photochemically stable and do not photobleach.16 Therefore, a UCP-based assay can be stored for a long time without a decrease in the light-emitting efficiency, can be read by repetitive scans, and can be archived for later verification. A number of instruments, based on fluorescence readout, have been reported for detection of hybridized targets.19 The most widely used imaging method in microarray analysis is a laserbased confocal microscope scanner, that detects the fluorophore emission corresponding to the band-pass filter utilized for each fluorophore.10,20,21 Recently, a two-laser hyperspectral microarray scanner has been developed for the multicolor microarray analysis.22 The hyperspectral imaging is able to identify multiple fluorescent dyes within a single scan. Because of the high cost of a confocal microscope scanner, development of new microarray detection methods is gaining more importance in the field of diagnostics. The instrumentation for the UCP fluorescence detection described in this report is simple, robust, and low-cost. Unlike confocal scanners, it can be constructed without any moving optical parts. This new anti-Stokes photoluminescence imaging device enables fluorescence readout and fluorescence imaging from solid phase, similar to conventional fluorescence measurement with continuous excitation, but gaining advantage of total background reduction. The instrument can be constructed using inexpensive infrared laser diode excitation, conventional optics, and a combination of long-pass and shortpass color glass and a band-pass emission filter. In this report, we describe a specific array-in-well test platform for robust adenovirus genotyping combining advantages of simple instrumentation and new UCP reporter technology. A desktop prototype device was constructed for multiplexed testing with laser diode excitation and a charge coupled device (CCD) image sensor. The whole procedure is carried out in microtiter wells, making it applicable to current laboratory practice. The feasibility of the assay concept was studied using a probe array detecting a selection of ten common adenovirus genotypes C01, C02, B03, E04, C05, C06, B07, B14, B21, and A31 causing human infections.

’ EXPERIMENTAL SECTION Adenovirus Samples and Controls. The prototypes of human adenovirus genotypes C01, C02, B03, E04, C05, C06, and B07 were originally obtained from the Centers for Disease Control and Prevention, Atlanta, USA. The prototypes C01, C02, B03, C05, and C06 were cultivated in the Hela cell culture (human cervix carcinoma cell-line Hela Ohio/Salisbury, Wiltshire, UK), and prototypes E04 and B07 were cultivated in the A549 cell culture (human lung carcinoma cell-line A549, American Type Culture Collection, ATCC, Rockville, Maryland, USA) for isolation of viral DNA. The cells were maintained in Eagle’s minimal essential medium (Gibco, Invitrogen, Carlsbad, California, USA). A recent isolate of hADV genotype B21 was kindly provided by HUSLAB, Helsinki, Finland. Nasopharyngeal specimens were selected from the processed clinical specimens sent to the Department of Virology, University of Turku, for respiratory virus antigen detection between December 2008 and March 2010. They had been tested positive for adenovirus hexon

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antigen using time-resolved fluoroimmunoassay and stored frozen at -20 C.23 Synthesis and Surface Modification of UCP Particles. The nanocrystalline NaYF4:Yb3þ,Er3þ materials (crystallite size 55 nm) were prepared with the coprecipitation method24 in the Laboratory of Materials Chemistry and Chemical Analysis, University of Turku, Finland as described earlier.25 These particles show upconversion luminescence in the 520-560 nm (green emission) and in the 650-700 nm (red emission) regions. The UCP particles were first suspended in 50% diethylene glycol (pH 5) and coated with a thin layer of silica through a silanization process in alkaline microemulsion, as described previously.26 In brief, phosphor particles were encapsulated with a uniform layer of silica using monomeric tetraethyl orthosilicate (Acros organics, Geel, Belgium), N-(3-trimethoxysilyl)propyl)ethylene diamine (Sigma-Aldrich, St. Louis, USA), and 3-(trihydroxysilyl)propyl methylphosphonate (Sigma-Aldrich). Amino groups on the surface of the particles were thereafter converted into carboxylic acid groups with glutaric anhydride (Sigma-Aldrich). Conjugation of Streptavidin to UCP Particles. The procedures for coating UCP particles with streptavidin have been described earlier.27 In brief, the carboxylic acid groups on the surface of the UCP particles were activated with EDC (1-ethyl3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinimide sodium salt) purchased from Fluka (Buchs, Switzerland). The UCP particles (10 mg/ mL) were conjugated with the streptavidin (2 mg/mL). The coupling reaction between the carboxylic acid groups of the UCP particles and the amino groups of streptavidin was stopped with excess of amino groups (50 mM glycine, pH 11). Quantitative Real-Time PCR and Sequencing. Nucleic acids were extracted from 200 μL aliquots of the specimens using NucliSens EasyMAG (bioMerieux, Boxtel, The Netherlands) or MagNA Pure (Roche, Mannheim, Germany) automated extractors with an elution volume of 55 or 50 μL, respectively, and the extracts were stored at -70 C. DNA was amplified using generic 50 end biotinylated ADHEX1R primer and ADHEX2F primer targeted to the hexon coding region (position 238-404), as described previously.28 The primers were used in an asymmetric quantitative polymerase chain reaction (qPCR) generating single-stranded target strand along with the double-stranded DNA. Reactions with melting curve analysis were carried out using Maxima SYBR Green qPCR Master Mix (Fermentas, St. Leon-Rot, Germany) in a RotorGene 6000 instrument (Corbett Research, Mortlake, Victoria, Australia). Briefly, amplification reaction was performed in a PCR mixture containing 0.1 μM of ADHEX2F primer and 0.45 μM of ADHEX1R primer. A 5 μL aliquot of DNA was added to a final volume of 25 μL. The cycling conditions consisted of denaturation and enzyme activation at 95 C for 15 min, 40 cycles of denaturation at 94 C for 15 s, annealing at 53 C for 60 s, and extension at 72 C for 45 s and final melting curve generation steps from 72 to 95 C with 1 C for 5 s increments. The resulting biotinylated amplicons were used unpurified in hybridizations or were purified (QuickClean, PCR Purification Kit, GenScript, USA) and eluted to a 30 μL volume of nucleasefree sterile water for sequencing. The amplicons were sequenced in the DNA Sequencing Service Laboratory of the Turku Centre for Biotechnology, Turku, Finland, in both directions using primers ADHEX2F and ADHEX1R. Oligonucleotide Microarrays. Oligonucleotide probes for the adenovirus prototype strains were designed using published 1457

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Table 1. Oligonucleotide Probes Used in the Arraya array position

a

sequence (50 f30 )

probe

position in the gene

1

probe-1

aminoC6-TCT TTG CTG GGC AAC GGT CGC TAC GTG C

283-307

2

probe-2

aminoC6-TTT TGT TGT TGG GAA ACG GCC GC

281-300

3

probe-3

aminoC6-TTC GCT GGT CTG CGC TAC AGG TCC ATG C

259-283

4

probe-4

aminoC6-TTT GCT ACG TGC CAT TCC ACA TCC AGG T

299-323

5

probe-5

aminoC6-TTC TCT CCT GCC GGG CTC ATA CAC CTA C

357-381

6

probe-6

aminoC6-TCT CTG GYC TAC GCT RCC GGT CCA TGC T

260-284

7

probe-7

aminoC6-TTC GCT TTT GGG CAA TGG TCG TTA CGT G

282-306

8 9

probe-8 probe-9

aminoC6-TCT ATC CAT GCT TTT GGG TAA CGG ACG T aminoC6-CCC GCT GCT GTT ACC AGG GTC CTA CAC T

276-300 354-378

10

probe-10

aminoC6-TTT CCC GCC ATG CGG GCC TGC GTT ACC G

251-275

11

probe-11

aminoC6-CCC TTT TGC CAT TAA GAA CCT CCT ACT CT

336-361

12

gADV1

aminoC6-TTT TTY AAC CAY CAC CGY AAY GCK GG

241-263

13

gADV2

aminoC6-TTT TTA ACC ACC ACC GCA ATG CTG G

242-263

Wobbles: K = T/G, Y = C/T.

Hexon sequences,4,26 aiming at a melting temperature around 70 C and a length of approximately 28 nucleotides (Table 1). The HPLC-purified oligonucleotides were obtained from Biomers.net GmbH (Ulm, Germany) and Thermo Fisher Scientific GmbH (Ulm, Germany). The probes were covalently immobilized at their 50 ends. To improve the probe availability for hybridization, the oligonucleotides were synthesized with three extra T/C nucleotides as spacers at their 50 ends before a terminal aminolink C6 group. Probes gADV1 (contains wobble bases) and gADV2 were used for the generic detection of human adenoviruses (Table 1). Each adenovirus genotype was aimed to be detected by the generic probes and by specific probes. Microarrays in transparent 96-well microtiter plates (Corning, Amsterdam, The Netherlands) were prepared at the VTT Medical Biotechnology Centre (Turku, Finland) utilizing a 50 -terminus amino group of the probe to bind oligonucleotides to the bottom of microtiter well using proprietary immobilization chemistry. Gibbs Free Energy Estimation. The predicted free energy change of the probe-target hybrid formation was calculated with the DINAMelt web server at http://dinamelt.bioinfo.rpi.edu/.29 Free energy change provides a thermodynamic measure of the affinity between a probe and a target, with more negative ΔG values being indicative of higher binding affinities. The calculations were performed using a two-state hybridization model, and perfectly matching duplexes were assumed. The values used were 60 C temperature, 2 nM total strand concentration, and 0.61 M Naþ concentration. Hybridization, Imaging and Image Analysis. Each PCR mixture was diluted 10-fold in denaturation solution (25 mM NaOH, 5 mM EDTA) and incubated for 10 min at room temperature, and thereafter, 25 μL was transferred as three replicates to the array wells containing 25 μL of a neutralization solution (100 mM Tris pH 7.75; 850 mM NaCl; 0.1% (v/v) Tween 20). The plate was incubated at 60 C for 1 h with shaking and washed (5 mM Tris pH 7.75; 150 mM NaCl; 0.05% (v/v) Tween 20) in a standard plate washer. Streptavidin-coated UCPs were diluted (0.005 mg/mL) in assay buffer containing 50 mM Tris-HCl, pH 7.8; 450 mM NaCl, 0.05% (w/v) NaN3; 0.5% (w/v) BSA; 0.01% (v/v) Tween-40; 0.05% (w/v) bovine gamma-globulin; and 20 μM diethylenetriaminepentaacetate. The solution containing streptavidin-coated UCPs was incubated with biotinylated samples on the array for 30 min at room temperature with shaking. The unbound label was washed, and

Figure 1. Setup of the anti-Stokes photoluminescence imaging device. The desktop prototype of the device is constructed for multiplexed testing based on laser diode excitation and the CCD image sensor.

the plate was dried. The assay was measured with a microplate reader modified for anti-Stokes photoluminescence imaging using infrared laser excitation and CCD detection. The images were saved as a 16-bit sif file format. The image analysis, spot detection, and quantification were performed with ImageJ software, version 1.43n (http://rsbweb.nih.gov/ij/index.html). Instrumentation. The array-in-well assay was measured with a Plate Chameleon microplate reader (Hidex Ltd., Turku, Finland) modified for the anti-Stokes photoluminescence imaging. A schematic diagram of the instrument is shown in Figure 1. The used radiation source was an infrared laser diode (DLS-976008; Optical Fiber Systems Inc., Massachusetts, USA) delivering 1458

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Analytical Chemistry 0.5-8.0 W of optical power at the wavelength of 976 ( 2 nm out of a small optical fiber (core diameter 100 μm). The laser fiber

Figure 2. Array-in-well test platform. Adenovirus genotype-specific 50 aminomodified probes were printed on microtiter wells. Denatured single-stranded 50 -biotinylated (1) sample oligonucleotides were hybridized to the genotype-specific oligonucleotide array for subtype determination. The detection was carried out with UCPs coated with streptavidin (2). Anti-Stokes photoluminescence was measured using a specific imaging device.

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was connected to a lens (F280FC-B; Thorlabs, M€unchen, Germany), which collimated the laser beam propagating out of the optical fiber. The laser beam passed through a 850 nm longpass filter (FRG-85012; UQG Optics Ltd., Cambridge, UK) and was reflected to the array well using a hot mirror (FM201; Thorlabs). The light from the array well was collected with a 50 mm camera lens (Nikkor AF 1:1.8 D; Nikon, Tokio, Japan), and the scattered laser radiation was rejected by two short-pass filters (005FG13-50 and 003FG13-50; Andover Corp., Salem, USA). The green emission of UCPs was detected via a 650 nm shortpass filter (E650SP-2P; Chroma Technology Corporation, Rockingham, USA), and the images were captured with a CCD camera (Andor Clara with Sony ICX285 CCD; Connecticut, USA) with a 50 mm objective lens (Nikkor AF 1:1.8 D; Nikon) using following parameters: 2 binning, 30 s exposure per well, and 7 W laser power. Data Analysis. An array well containing all the reagents except the sample was used as a blank background. Instrument background was subtracted from the blank background and spot signal intensities. Specific spot intensity was determined as the mean intensity of the pixels within the spot minus the mean intensity of the pixels within the blank background. For each specific spot, signal-to-background ratios (mean specific spot intensity/mean intensity of blank background) were obtained.

Figure 3. Hybridization patterns of the prototype adenovirus genotypes C01, C02, B03, E04, C05, C06, B07, and B21 to the array. The probe number does not refer to adenovirus type. For each prototype, the combination of responses results in a unique pattern. The pattern can be thought of as a “bar code” response to the specific signature of each strain hybridized to the array (in array layout marked green probes). Samples were hybridized also to control spots 12 and 13 which are not marked in the array layout. 1459

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’ RESULTS AND DISCUSSION Assay Theory. The array-in-well test platform is illustrated in Figure 2. It is not always possible to design a single probe that would perfectly match within the genotype, but utilizing microarray technology, it is feasible to increase the number of probes to identify one genotype.8,30 The length of probes typically used in printed oligonucleotide arrays range from 25 to 80 mer. In general, long probes have higher sensitivity but lower specificity than short probes.31 In this work, eleven adenovirus genotypespecific 50 -aminomodified probes were printed covalently in triplicate on microtiter wells. Probes were designed to recognize selected adenovirus genotypes C01, C02, B03, E04, C05, C06, B07, B14, B21, and A31. In this study, the capture probes contained three additional T/C nucleotides at the 50 end that

Figure 4. Mean hybridization signals of the adenovirus prototype strains plotted as a function of the predicted thermodynamic strengths of the probes. The predicted free energy change calculations were performed as described in Experimental Section. The calculation of the correlation coefficient R was performed using Origin v8.0773.

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could be utilized to increase the hybridization efficiency by functioning as a spacer.32 The oligonucleotide spots were hybridized with the denatured biotinylated PCR product using streptavidin-coated UCPs as labels. The assay was measured from solid phase with the new anti-Stokes photoluminescence imager using infrared laser excitation and CCD detection. Array-in-Well Assay Development. Optimization of the assay conditions were done with synthesized single-stranded 50 -biotinylated target oligonucleotides (data not shown) and asymmetric qPCR amplified products of cultured adenovirus genotypes C01, C02, B03, E04, C05, C06, B07 and B21. The adenovirus genotypes B14 and A31 were not available. The sensitivity of the asymmetric qPCR was determined with adenovirus C02 genotype that was detected at concentrations of less than 10 copies per qPCR reaction. Asymmetric qPCR detected nucleic acids from all the prototypes and clinical samples used in the study (treshold values 7-29). The eleven spotted probes in the bottom of a microtiter well gave clear hybridization patterns to the adenovirus genotypes C01, C02, B03, E04, C05, C06, B07, and B21 (Figure 3). Samples were sequenced to confirm the obtained DNA-typing results. Our data showed a clear correlation (R = 0.74) between the hybridization intensity and thermodynamic strengths (ΔG) of the oligonucleotides (Figure 4). As we have previously shown, the calculated ΔG can be used to obtain a useful empirical rule for the behavior of oligonucleotide microarray probes.8 In the present setup, a predicted ΔG of approximately -14 kcal/mol or lower was required for detection, and a value lower than -16 kcal/mol was required for strongly positive signals. The correlation between the intensity and ΔG can be used in the quality control of the microarray hybridizations. The UCP particles were not binding significantly to the nonspecific probe spots (signals between 680 and 3000) or to the surrounding area of spots (signals between 660 and 1600). The detected instrument background signal was around 500. The actual specific intensity values for the adenovirus prototypes were

Figure 5. Fluorescence image of the array-in-well assay result for samples A-F. Comparison to Figure 3 indicates clear ability to assign the samples A, B, C, D, E, and F to B03, C02, C06, C01, C05, and E04, respectively. 1460

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Analytical Chemistry between 12 000 and 35 000 and the signal-to-background ratios of detected specific signals were from 179 to 545. The detected hybridization signals between the samples and the control probe gADV2 (probe-13) were generally higher than signals with gADV1 (probe-12). Validation of the Array-in-Well Assay with Clinical Samples. The assay was validated on a total of 55 clinical nasopharyngeal specimens that previously tested positive for adenovirus by time-resolved fluoroimmunoassay. The array-in-well assay detected the presence of six different adenovirus genotypes among the clinical sample panel (Figure 5). Randomly selected samples were sequenced to ensure the assay result. In all cases, the results of genotype obtained by the microarray assay matched with those obtained by sequencing. The microarray provided a clear hybridization pattern for each clinical sample. The genotype C02 showed the highest prevalence, accounting for 25.5% of the samples. Representatives of other species were observed less commonly: genotype C01 in 18.2%; genotype B03 in 21.8%; genotype E04 in 14.5%; genotype C05 in 7.3%; and genotype C06 in 12.7%.

’ CONCLUSIONS The developed array-in-well approach utilizing upconverting phosphor reporter technology and the novel instrumentation for UCP imaging will be applicable in various multianalyte studies. UCP technology enables a simple and low-cost readout system and less background signal than many other widely used approaches. On the basis of the results of the array-in-well assay, high-throughput and specific detection of adenovirus genotype from clinical nasopharyngeal specimens can be performed as described in the present study. The identification of individual hAdV genotypes is based on quantitative real-time PCR amplification of a part of conserved hexon gene region and subsequent genotype analysis of the amplified PCR fragment by the array-inwell assay.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: minna.yliharsila@utu.fi. Tel: þ358-2-333-8067. Fax: þ358-2-333-8050.

’ ACKNOWLEDGMENT The assistance of Riikka Arppe for preparing streptavidin coated UCPs used in this study is gratefully acknowledged. This work was supported by DIA-NET, the Graduate School of Advanced Diagnostic Technologies and Applications, and Tekes, the Finnish Funding Agency for Technology and Innovation. ’ REFERENCES

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