Vox Sanguinis (2015) 108, 197–204 © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12207
ORIGINAL PAPER
PCR-free blood group genotyping using a nanobiosensor D. Brouard,1 O. Ratelle,2 J. Perreault,1 D. Boudreau2 & M. St-Louis1 1
Recherche et de veloppement, He ma-Que bec, Que bec, QC, Canada De partement de chimie et Centre d’optique, photonique et laser (COPL), Universite Laval, Que bec, QC, Canada
2
Background and Objectives The last two decades have seen major developments in genotyping assays to facilitate the procurement of red blood cell units to alloimmunized patients. To make genotyping faster, simpler and less costly, a nanotechnology approach based on metal/silica fluorescent nanoparticles and a polymer-based hybridization optical transducer was designed. The objectives of this study were (1) to verify whether this nanobiosensor has the ability to discriminate single nucleotide polymorphisms in non-amplified genomic DNA and (2) to establish whether the signal generated by the nanobiosensor is sufficiently intense to be detected by standard flow cytometry. Materials and Methods Silver-core silica-shell fluorescent nanoparticles (Ag@SiO2) were prepared, and amine-modified DNA probes were grafted to their surface. A cationic conjugated polymer was electrostatically bound to the surface probes to become optically active upon hybridization with a target. Two nanobiosensor formulations specific to DO*01 and DO*02 alleles were prepared. DNA was extracted from whole blood and mixed with the nanobiosensor for hybridization. The nanobiosensor fluorescence was measured by flow cytometry. Results Nine volunteers were typed for Dombrock blood group antigens DO*01 and DO*02. A statistically significant increase in the optical transduction signal was observed for sequence-specific samples. All nine genotypes were correctly identified when compared to standardized PCR assays.
Received: 5 June 2014, revised 21 July 2014, accepted 16 September 2014, published online 3 December 2014
Conclusion The nanobiosensor provides rapid and simple genotyping of blood group antigens from unamplified genomic DNA and can be measured using standard flow cytometers. This PCR-free approach could be applied to any known genetic polymorphism. Key words: composite nanoparticles, core-shell nanoparticles, DNA genotyping, flow cytometry, fluorescent nanoparticles, PCR-free genotyping.
Introduction The classical method for blood typing, haemagglutination, owes its popularity to good specificity and sensitivity, as well as being simple, fast and requiring basic Correspondence: Danny Brouard and Maryse St-Louis, Hema-Quebec, Recherche et developpement, 1070, avenue des Sciences-de-la-Vie, Quebec (Quebec) G1V 5C3, Canada E-mail: [email protected] and [email protected] and Denis Boudreau, Departement de chimie et Centre d’optique, photonique et laser (COPL), Universite Laval, Quebec (Quebec) G1V 0A6, Canada. E-mail: [email protected]
equipment. However, with the growing need for compatible blood, it is becoming difficult to rely on serological methods alone to handle polymorphisms. They require reliable antisera, are labour-intensive and present major limitations with recently transfused patients, RBCs coated with IgG and the discrimination of some zygosity (RHD in D-positive individuals). On the other hand, blood grouping by molecular biology methods has been attracting increasing attention in the field of transfusion medicine [1, 2]. This interest is mainly attributed to the increased knowledge of alleles responsible for differences in blood group antigens [3, 4]. In fact, there are 33 blood group systems and more than 300 antigens currently identified [5].
197
198 D. Brouard et al.
To better answer the need for compatible blood products, rapid and accurate methods are essential to define the blood group status of a large number of individuals. The challenge is impressive since a majority of genetic differences between blood groups are caused by a single variation known as single nucleotide polymorphism (SNP) [6]. SNP detection strategies such as PCR-restriction fragment length polymorphism (PCR-RFLP) [7], sequence-specific primer-PCR (SSP-PCR) [8], multiplex PCR [9], realtime PCR [10], Sanger DNA sequencing and pyrosequencing, among others, prove to be just as reliable as serology. More recently, commercially available highthroughput integrated systems have shown superb results in terms of detection accuracy, specificity and sensitivity [11–15]. However, despite their enviable qualities, genotyping strategies involving enzymatic amplification are known to be prone to contamination or enzyme inhibition and are time consuming or costly, or both [16–18]. In the case of high-throughput techniques, it may take a full day before results are released and the economy of scale expected from this technology is reached only for batches of hundreds of samples, which is less than ideal when smaller sample sets must be analysed, for example for genotyping transfusion recipients in a hospital setting. Lower throughput assays such as real-time PCR can be performed for single samples and a single antigen with DNA-to-answer results available in 90 min to 2 h, but the procedures require several steps performed by skilled personnel. Because of these factors, blood group genotyping is still generally confined to particular situations such as rare blood groups, weakly expressed antigens, patients with multiple transfusions and whenever reagents used in haemagglutination techniques are too costly, unreliable or simply not available. There is therefore a need for a faster, simpler and more cost-effective genotyping technology working in a ‘sample-to-answer’ mode with a minimum number of preparation steps. One such strategy is to amplify the signal generated by the recognition of hybridization events between oligonucleotide probes and perfectly complementary targets rather than amplifying the number of targets by PCR. Nanomaterials possess very unique properties that can benefit the design of sensing or imaging DNA devices. For example, the change in colour of a biosensor associated with changes in distance between gold nanoparticles (NP) has been used for sensing applications [19]. Other examples include surface-enhanced Raman spectroscopy (SERS) or fluorescence with metal nanoparticles to monitor gene expression in cancer cells without enzymatic amplification [20, 21]. The use of quantum dots or other luminescent nanoparticles to amplify the signal generated by DNA hybridization for gene and SNP detection
applications has also been reported [22, 23]. Cationic conjugated polymers (CCP), which often display different colours or other optical properties when associated with particular biomolecules, such as DNA or proteins, were investigated as transducers of DNA hybridization [24]. Notably, Ho et al. designed a DNA sensing scheme based on the changes in fluorescence intensity of a CCP upon association with either single-strand (ssDNA) or doublestrand DNA (dsDNA) [25]. When bound to ssDNA, the polymer (a polythiophene derivative) adopts a conformation in which fluorescence is quenched. When perfectly complementary ssDNA is added, hybridization with the probe forces the polymer to change configuration and recover its intrinsic fluorescence [26]. The detection sensitivity of this ‘switched-on’ biosensor was later improved by labelling the ssDNA probes with an acceptor fluorophore to establish resonant energy transfer (RET) between the polymer donor and the fluorophore acceptor, which resulted in a fluorescence signal increase relative to the fluorescence of the polymer alone [27]. Unfortunately, the self-assembled nature of the aggregated structure needed to achieve proximity between CCP and acceptor lacked reproducibility and robustness. To overcome these limitations, an approach based on hybrid metal/silica nanoparticles incorporating an acceptor dye and grafted with capture probes was designed (Fig. 1a) [28]. As in the CCP-based sensing scheme described above, probe-target hybridization switches on the CCP fluorescence, with the RET occurring in this case between the CCP donor and the nanoparticle-bound acceptor fluorophore. It has been shown previously that the metal core in these nanocomposites enhances the brightness and photostability of the donor and acceptor fluorophores while increasing the range of RET between them [29, 30]. It has also been shown that the addition of the cationic conjugated polymer to the probe-grafted nanoparticles neutralizes the negatively charged surface of the latter and leads to the creation of nanoparticle aggregates [30]. This aggregation process enables collective interactions between nanoparticles, enhancing the local electric field and the overall fluorescence intensity of recognition events. A detection limit of 1 9 104 copies was previously reported with this nanobiosensor using short 20-mer oligonucleotides as model analytes [28, 31], and its use was recently demonstrated for the direct molecular detection of the SRY gene from unamplified genomic DNA extracted from blood samples with nine samples of 10 being correctly identified [31]. However, the analytical capabilities of this nanobiosensor had not, until now, been evaluated for SNP detection and blood group genotyping applications. The goal of this study was to demonstrate the ability of the nanobiosensor to perform blood group genotyping © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 197–204
PCR-free blood group genotyping 199
(a)
(b)
Fig. 1 (a) Fluorescent core-shell nanoparticles (Ag@SiO2) are made of a 50-nm diameter silver core and a 10-nm thick eosin doped silica shell. They are functionalized with oligonucleotide probes (‘probe-grafted Ag@SiO20 ), and the polymer transducer is then added to electrostatically adsorb to the negatively charged probes and form nanobiosensor probes. Hybridization with oligonucleotide targets or genomic DNA activates the transducer and allows coupling of a suitable excitation source to the luminescent core-shell nanoparticles with enhanced brightness and photostability. (b) Transmission electronic microscopy photograph of Ag@SiO2 core-shell nanoparticles.
applications directly from unamplified DNA samples by flow cytometry. Silver/silica nanoparticles (Ag@SiO2) were functionalized with capture probes specific to DO*01 and DO*02 alleles and were used to determine the probable Doa and Dob phenotype of nine individuals.
Materials and methods Reagents All oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA) in a standard desalting purification grade, except for the Cy5.5-labelled probes and targets, which were HPLC purified. Sterile water was purchased from Sigma Aldrich (St-Louis, MO, USA). PCR reagents were all purchased from Life Technologies (Burlington, ON, Canada): 109 PCR Buffer II, MgCl2, AmpliTaq Gold DNA polymerase and dNTPs.
Preparation of the nanobiosensor The nanobiosensor was prepared following the protocol described in the supplementary file. To summarize, coreshell nanoparticles having an average core diameter of 50 – 10 nm and a silica shell thickness of 10 – 1 nm (Fig. 1b) were synthesized via the reduction of silver nitrate by citrate ions followed by a sol-gel silica condensation step. The RET acceptor fluorophore (eosin) was covalently incorporated in the silica shell and ssDNA probes specific to either DO*01 or DO*02 alleles (793A > G) (see Table 1 for base sequences) were then grafted to the nanoparticles’ surface. Probe-grafted fluorescent core-shell nanoparticles obtained in this manner are very stable and can be stored for at least 3 months at 4°C in the dark without any observed loss of brightness, © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 197–204
Table 1 Oligonucleotide probe sequences used in the fabrication of the nanobiosensor ID
Sequence
DO*01 probe
50 -/AmMC12/ACC ACC CAA GAG GAG ACT GGT TGC AGT TG -30 50 -/AmMC12/ACC ACC CAA GAG GAA ACT GGT TGC AGT TG -30 50 -/AmMC12/ACC ACC CAA GAG GAG ACT GGT TGC AGT TG/Cy5.5/-30
DO*02 probe DO*01 Cy5.5 probe
with lot-to-lot variations in signal output
ORIGINAL PAPER
PCR-free blood group genotyping using a nanobiosensor D. Brouard,1 O. Ratelle,2 J. Perreault,1 D. Boudreau2 & M. St-Louis1 1
Recherche et de veloppement, He ma-Que bec, Que bec, QC, Canada De partement de chimie et Centre d’optique, photonique et laser (COPL), Universite Laval, Que bec, QC, Canada
2
Background and Objectives The last two decades have seen major developments in genotyping assays to facilitate the procurement of red blood cell units to alloimmunized patients. To make genotyping faster, simpler and less costly, a nanotechnology approach based on metal/silica fluorescent nanoparticles and a polymer-based hybridization optical transducer was designed. The objectives of this study were (1) to verify whether this nanobiosensor has the ability to discriminate single nucleotide polymorphisms in non-amplified genomic DNA and (2) to establish whether the signal generated by the nanobiosensor is sufficiently intense to be detected by standard flow cytometry. Materials and Methods Silver-core silica-shell fluorescent nanoparticles (Ag@SiO2) were prepared, and amine-modified DNA probes were grafted to their surface. A cationic conjugated polymer was electrostatically bound to the surface probes to become optically active upon hybridization with a target. Two nanobiosensor formulations specific to DO*01 and DO*02 alleles were prepared. DNA was extracted from whole blood and mixed with the nanobiosensor for hybridization. The nanobiosensor fluorescence was measured by flow cytometry. Results Nine volunteers were typed for Dombrock blood group antigens DO*01 and DO*02. A statistically significant increase in the optical transduction signal was observed for sequence-specific samples. All nine genotypes were correctly identified when compared to standardized PCR assays.
Received: 5 June 2014, revised 21 July 2014, accepted 16 September 2014, published online 3 December 2014
Conclusion The nanobiosensor provides rapid and simple genotyping of blood group antigens from unamplified genomic DNA and can be measured using standard flow cytometers. This PCR-free approach could be applied to any known genetic polymorphism. Key words: composite nanoparticles, core-shell nanoparticles, DNA genotyping, flow cytometry, fluorescent nanoparticles, PCR-free genotyping.
Introduction The classical method for blood typing, haemagglutination, owes its popularity to good specificity and sensitivity, as well as being simple, fast and requiring basic Correspondence: Danny Brouard and Maryse St-Louis, Hema-Quebec, Recherche et developpement, 1070, avenue des Sciences-de-la-Vie, Quebec (Quebec) G1V 5C3, Canada E-mail: [email protected] and [email protected] and Denis Boudreau, Departement de chimie et Centre d’optique, photonique et laser (COPL), Universite Laval, Quebec (Quebec) G1V 0A6, Canada. E-mail: [email protected]
equipment. However, with the growing need for compatible blood, it is becoming difficult to rely on serological methods alone to handle polymorphisms. They require reliable antisera, are labour-intensive and present major limitations with recently transfused patients, RBCs coated with IgG and the discrimination of some zygosity (RHD in D-positive individuals). On the other hand, blood grouping by molecular biology methods has been attracting increasing attention in the field of transfusion medicine [1, 2]. This interest is mainly attributed to the increased knowledge of alleles responsible for differences in blood group antigens [3, 4]. In fact, there are 33 blood group systems and more than 300 antigens currently identified [5].
197
198 D. Brouard et al.
To better answer the need for compatible blood products, rapid and accurate methods are essential to define the blood group status of a large number of individuals. The challenge is impressive since a majority of genetic differences between blood groups are caused by a single variation known as single nucleotide polymorphism (SNP) [6]. SNP detection strategies such as PCR-restriction fragment length polymorphism (PCR-RFLP) [7], sequence-specific primer-PCR (SSP-PCR) [8], multiplex PCR [9], realtime PCR [10], Sanger DNA sequencing and pyrosequencing, among others, prove to be just as reliable as serology. More recently, commercially available highthroughput integrated systems have shown superb results in terms of detection accuracy, specificity and sensitivity [11–15]. However, despite their enviable qualities, genotyping strategies involving enzymatic amplification are known to be prone to contamination or enzyme inhibition and are time consuming or costly, or both [16–18]. In the case of high-throughput techniques, it may take a full day before results are released and the economy of scale expected from this technology is reached only for batches of hundreds of samples, which is less than ideal when smaller sample sets must be analysed, for example for genotyping transfusion recipients in a hospital setting. Lower throughput assays such as real-time PCR can be performed for single samples and a single antigen with DNA-to-answer results available in 90 min to 2 h, but the procedures require several steps performed by skilled personnel. Because of these factors, blood group genotyping is still generally confined to particular situations such as rare blood groups, weakly expressed antigens, patients with multiple transfusions and whenever reagents used in haemagglutination techniques are too costly, unreliable or simply not available. There is therefore a need for a faster, simpler and more cost-effective genotyping technology working in a ‘sample-to-answer’ mode with a minimum number of preparation steps. One such strategy is to amplify the signal generated by the recognition of hybridization events between oligonucleotide probes and perfectly complementary targets rather than amplifying the number of targets by PCR. Nanomaterials possess very unique properties that can benefit the design of sensing or imaging DNA devices. For example, the change in colour of a biosensor associated with changes in distance between gold nanoparticles (NP) has been used for sensing applications [19]. Other examples include surface-enhanced Raman spectroscopy (SERS) or fluorescence with metal nanoparticles to monitor gene expression in cancer cells without enzymatic amplification [20, 21]. The use of quantum dots or other luminescent nanoparticles to amplify the signal generated by DNA hybridization for gene and SNP detection
applications has also been reported [22, 23]. Cationic conjugated polymers (CCP), which often display different colours or other optical properties when associated with particular biomolecules, such as DNA or proteins, were investigated as transducers of DNA hybridization [24]. Notably, Ho et al. designed a DNA sensing scheme based on the changes in fluorescence intensity of a CCP upon association with either single-strand (ssDNA) or doublestrand DNA (dsDNA) [25]. When bound to ssDNA, the polymer (a polythiophene derivative) adopts a conformation in which fluorescence is quenched. When perfectly complementary ssDNA is added, hybridization with the probe forces the polymer to change configuration and recover its intrinsic fluorescence [26]. The detection sensitivity of this ‘switched-on’ biosensor was later improved by labelling the ssDNA probes with an acceptor fluorophore to establish resonant energy transfer (RET) between the polymer donor and the fluorophore acceptor, which resulted in a fluorescence signal increase relative to the fluorescence of the polymer alone [27]. Unfortunately, the self-assembled nature of the aggregated structure needed to achieve proximity between CCP and acceptor lacked reproducibility and robustness. To overcome these limitations, an approach based on hybrid metal/silica nanoparticles incorporating an acceptor dye and grafted with capture probes was designed (Fig. 1a) [28]. As in the CCP-based sensing scheme described above, probe-target hybridization switches on the CCP fluorescence, with the RET occurring in this case between the CCP donor and the nanoparticle-bound acceptor fluorophore. It has been shown previously that the metal core in these nanocomposites enhances the brightness and photostability of the donor and acceptor fluorophores while increasing the range of RET between them [29, 30]. It has also been shown that the addition of the cationic conjugated polymer to the probe-grafted nanoparticles neutralizes the negatively charged surface of the latter and leads to the creation of nanoparticle aggregates [30]. This aggregation process enables collective interactions between nanoparticles, enhancing the local electric field and the overall fluorescence intensity of recognition events. A detection limit of 1 9 104 copies was previously reported with this nanobiosensor using short 20-mer oligonucleotides as model analytes [28, 31], and its use was recently demonstrated for the direct molecular detection of the SRY gene from unamplified genomic DNA extracted from blood samples with nine samples of 10 being correctly identified [31]. However, the analytical capabilities of this nanobiosensor had not, until now, been evaluated for SNP detection and blood group genotyping applications. The goal of this study was to demonstrate the ability of the nanobiosensor to perform blood group genotyping © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 197–204
PCR-free blood group genotyping 199
(a)
(b)
Fig. 1 (a) Fluorescent core-shell nanoparticles (Ag@SiO2) are made of a 50-nm diameter silver core and a 10-nm thick eosin doped silica shell. They are functionalized with oligonucleotide probes (‘probe-grafted Ag@SiO20 ), and the polymer transducer is then added to electrostatically adsorb to the negatively charged probes and form nanobiosensor probes. Hybridization with oligonucleotide targets or genomic DNA activates the transducer and allows coupling of a suitable excitation source to the luminescent core-shell nanoparticles with enhanced brightness and photostability. (b) Transmission electronic microscopy photograph of Ag@SiO2 core-shell nanoparticles.
applications directly from unamplified DNA samples by flow cytometry. Silver/silica nanoparticles (Ag@SiO2) were functionalized with capture probes specific to DO*01 and DO*02 alleles and were used to determine the probable Doa and Dob phenotype of nine individuals.
Materials and methods Reagents All oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA) in a standard desalting purification grade, except for the Cy5.5-labelled probes and targets, which were HPLC purified. Sterile water was purchased from Sigma Aldrich (St-Louis, MO, USA). PCR reagents were all purchased from Life Technologies (Burlington, ON, Canada): 109 PCR Buffer II, MgCl2, AmpliTaq Gold DNA polymerase and dNTPs.
Preparation of the nanobiosensor The nanobiosensor was prepared following the protocol described in the supplementary file. To summarize, coreshell nanoparticles having an average core diameter of 50 – 10 nm and a silica shell thickness of 10 – 1 nm (Fig. 1b) were synthesized via the reduction of silver nitrate by citrate ions followed by a sol-gel silica condensation step. The RET acceptor fluorophore (eosin) was covalently incorporated in the silica shell and ssDNA probes specific to either DO*01 or DO*02 alleles (793A > G) (see Table 1 for base sequences) were then grafted to the nanoparticles’ surface. Probe-grafted fluorescent core-shell nanoparticles obtained in this manner are very stable and can be stored for at least 3 months at 4°C in the dark without any observed loss of brightness, © 2014 International Society of Blood Transfusion Vox Sanguinis (2015) 108, 197–204
Table 1 Oligonucleotide probe sequences used in the fabrication of the nanobiosensor ID
Sequence
DO*01 probe
50 -/AmMC12/ACC ACC CAA GAG GAG ACT GGT TGC AGT TG -30 50 -/AmMC12/ACC ACC CAA GAG GAA ACT GGT TGC AGT TG -30 50 -/AmMC12/ACC ACC CAA GAG GAG ACT GGT TGC AGT TG/Cy5.5/-30
DO*02 probe DO*01 Cy5.5 probe
with lot-to-lot variations in signal output
