Jing Zhu‡a,
Chunmei Qiu‡b,
Mirkó Pallaab,
ThaiHuu Nguyena,
James J. Russob,
Jingyue Jub and
Qiao Lin*a
aDepartment of Mechanical Engineering, Columbia University, New York, NY 10027, USA. E-mail: qlin@columbia.edu; Tel: +1-212-854-1906
bDepartment of Chemical Engineering, Columbia University, New York, NY, USA 10027
First published on 7th November 2013
Single-nucleotide polymorphisms (SNPs) are the most abundant type of genetic variations; they provide the genetic fingerprint of individuals and are essential for genetic biomarker discoveries. Accurate detection of SNPs is of great significance for disease prevention, diagnosis and prognosis, and for prediction of drug response and clinical outcomes in patients. Nevertheless, conventional SNP genotyping methods are still limited by insufficient accuracy or labor-, time-, and resource-intensive procedures. Microfluidics has been increasingly utilized to improve efficiency; however, the currently available microfluidic genotyping systems still have shortcomings in accuracy, sensitivity, throughput and multiplexing capability. To address these challenges, we developed a multi-step SNP genotyping microfluidic device, which performs single-base extension of SNP specific primers and solid-phase purification of the extension products on a temperature-controlled chip. The products are ready for immediate detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), providing identification of the alleles at the target loci. The integrated device enables efficient and automated operation, while maintaining the high accuracy and sensitivity provided by MS. The multiplex genotyping capability was validated by performing rapid, accurate and simultaneous detection of 4 loci on a synthetic template. The microfluidic device has the potential to perform automatic, accurate, quantitative and high-throughput assays covering a broad spectrum of applications in biological and clinical research, drug development and forensics.
A variety of biological methods have been developed for SNP genotyping, including the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP),5 DNA hybridization,6 TaqMan,7 allele-specific ligation8 and allele-specific single-base extension (SBE),9 using detection schemes such as fluorescence detection and mass spectrometry (MS). The introduction of micro- and nanotechnology has revolutionized biological analysis, as the miniaturization of assays facilitates integration, improves speed, efficiency and accuracy, reduces labor, and provides the potential for high-throughput and point-of-care applications. Furthermore, the use of smaller sample volumes lowers reagent consumption and energy requirements, and shortens reaction cycles.10,11 Integrated with micro- and nanotechnology, a variety of genotyping microdevices have been explored. For example, the allele-specific hybridization and ligation protocols with fluorescent detection have been incorporated into a microchip for the determination of influenza virus subtypes,12 and for the discrimination of single-nucleotide mismatches.13,14 The RFLP-based microchip combined with a capillary electrophoresis separation device has also been developed to identify SNPs in the thiopurine S-methyltransferase gene.15 In addition, the TaqMan genotyping assay has been integrated into nanofluidic circuits and commercialized by Fluidigm Corporation with significant advantages over the conventional thermal cycling process.16,17 Nevertheless, optical detection has its intrinsic limitations, as optical signals for the use of detecting ultra-low abundance nucleotide variations tend to generate false-positive results, causing insufficient accuracy and sensitivity. Mass spectrometric detection is advantageous over optical detection in terms of sensitivity and accuracy, which are especially critical for detecting low frequency mutations, such as somatic mutations in tumors and mitochondrial mutations.18,19 However, the only commercially available mass spectrometric genotyping platform, the Sequenom MassARRAY®, is not integrated into an automated system, which requires substantial manual labor prior to mass spectrometric analysis, such as traditional thermocycling and manual resin purification. The manual handling of each step would not only contribute to errors, but also limit the throughput. Moreover, its multiplexing capability is also limited, as unextended primers remain in the final product and potentially overlap with extended primers targeting different polymorphic sites.20 This limitation can be overcome by introducing solid-phase purification (SPP), in which only extended primers are captured for mass spectrometric analysis, eliminating interference from the excess primers.21 Therefore, it is highly desirable to integrate SBE and SPP required for uniplex or multiplex SNP genotyping in a single microfluidic device, so that samples can be processed automatically while maintaining the high accuracy and sensitivity of MS.
To achieve this goal, we previously reported a mass spectrometric SNP genotyping method using microfluidic bead-based SBE.22 This paper further presents the development and testing of a multi-step SNP genotyping microfluidic device that additionally includes a two-step solid-phase purification scheme for solution-based SBE product, making multiplexing practically attainable. All required steps of the SNP genotyping reactions are integrated in the device, which is coupled to external instrumentation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Experiments with uniplex and 4-plex SNP detection have demonstrated that the device is capable of accurate, rapid and automated analysis with reduced consumption of samples and reagents, and can potentially be used in high-throughput and multiplex SNP genotyping.
The microfluidic genotyping device is fabricated using standard MEMS technology. Briefly, metal (Cr and Au) thin films are deposited, patterned and passivated to form the micro heaters and temperature sensors on a glass chip. Next, the polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning Inc., Midland, MI) sheet bearing microfluidic features is fabricated via soft lithography, and then bonded to a thin PDMS membrane that is spin-coated onto another silicon mold defining oil-filled channels for microvalve actuation. The resulting PDMS stack is bonded to the temperature control chip, and the inner surfaces of the SBE microchamber are coated with Parylene C. Finally, streptavidin beads and C18 bonded silica microspheres are packed in the SPP chamber and desalting channel, respectively (Fig. 2B). An image of a fabricated device is shown in Fig. 2C. The detailed microfabrication procedure is shown in ESI (Fig. S1†).
Fluid control is achieved using microfabricated pressure-driven valves.25 Four oil-filled channels, each controlled by an air control valve (6464K16, McMaster-Carr, NJ), are connected to a nitrogen gas tank (Tech Air, NY) via a pressure regulator (CONCOA North America, VA). The microfluidic device inlets are connected to a set of five syringes that contain 50% acetonitrile (ACN), 0.1 M triethylammonium acetate buffer (TEAA), binding and washing (B&W) buffer (5 mM Tris–HCl, 0.5 mM EDTA, 1 M NaCl, and 0.01% Tween 20, pH 7.5), 100 mM TCEP (pH 9.0, adjusted with ammonium hydroxide) and DI water respectively; and the fluidic flow is regulated by syringe pumps (KD210P, KD Scientific, MA, and NE-1000, New Era Pump System, NY). The outlet is connected to a microcentrifuge tube to collect the final genotyping product for MALDI-TOF MS analysis (Fig. 3).
To conduct the microfluidic SNP genotyping, the C18 silica microspheres and streptavidin beads are first rinsed to provide an optimal binding condition. Then, the sample with target template and SBE reagent is introduced into the SBE chamber and goes through 30 thermal cycles of 94 °C for 15 s, 40 °C for 60 s and 69 °C for 30 s. Next, the SBE product is transferred into the SPP chamber and extracted under continuous flow conditions. Then, the primer extension product is released from streptavidin beads by cleaving the linker with 100 mM TCEP (pH 9.0). Finally, the cleaved and purified primer extension product is transferred to the desalting channel, where it is desalted and eluted with 2 μL of 50% ACN, and finally characterized with a Voyager DE™ MALDI-TOF mass spectrometer (Applied Biosystems®, Life Technologies, Grand Island, NY). This experimental procedure is described in detail in ESI (Fig. S3 & S4†).
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Fig. 4 MALDI-TOF mass spectrum of SBE product (the peak marked with an asterisk is presumably due to the impurities in the commercial synthetic primer). |
In another optimization experiment, we evaluated the effect of flow rate on SPP efficiency. Approximately 170 pmol biotinylated ssDNA in 5 μL 1× Thermo Sequenase™ reaction buffer was introduced into the SPP microchambers at varying flow rates of 0.2 μL min−1, 1 μL min−1, 5 μL min−1, and 20 μL min−1 respectively, followed by DI water wash. After SPP, the total amounts of biotinylated ssDNA not captured by the beads for both flow rates of 0.2 μL min−1 and 1 μL min−1 were below 2 pmol (Fig. 5B). However, at flow rates of 5 μL min−1 and 20 μL min−1, the amounts of uncaptured biotinylated ssDNA fragments increased to about 5 pmol and 13 pmol (Fig. 5B), respectively. These results imply the binding capacity of streptavidin beads packed in the SPP microchamber decreases as the flow rate increases, since the reaction time between biotin and streptavidin is reduced. Nevertheless, taking into account the operation time, 5 μL min−1 with 5 pmol loss in a total of 170 pmol is still considered an efficient binding condition.
Finally, in order to evaluate the binding specificity of streptavidin to the biotinylated DNA products, approximately 140 pmol of unbiotinylated ssDNA (5163: 5′-GATAGGACTCATCACCA-3′) in 5 μL 1× Thermo Sequenase™ reaction buffer was flowed into the SPP microchamber packed with streptavidin beads at 1 μL min−1, followed by the same DI water wash (Fig. 5C). The amount of ssDNA collected was about 126 pmol, which indicated a negligible non-specific binding of unbiotinylated ssDNA to the streptavidin beads. At faster flow rates, the non-specific binding would be even less. Given these results, we conclude that performing SPP at 5 μL min−1 is both binding- and time-efficient. At this rate, the extraction of 10 μL reaction solution could be simply achieved within 2 min, as compared to in-tube reaction, which requires constant mixing and longer reaction time.26 The microfluidic SPP device also improves sample recovery, as the physical entrapment of the beads allows extensive washing and provides a more efficient SPP process.
To characterize DNA recovery from the solid phase, the same SBE reactions were performed as described in Section 4.2 using a commercial thermal cycler (Eppendorf Mastercycler® Personal). Similarly to our previous experiments, the SBE extension products (6180 Da) were purified by streptavidin microbeads packed in the SPP microchamber. Then, 10 μL of 100 mM TCEP solution (pH 9.0) was introduced and incubated at 65 °C for 10 min. Incubation of the extension products in TCEP solution resulted in the cleavage of the linker tethering the biotin to the dideoxyribonucleotides. Theoretically, the mass of cleaved product can be calculated by the equation mp = me − mc, where mp is the mass of cleaved product, me is the mass of extended primer and mc is the mass change upon cleavage (476 Da). As shown in Fig. 5D, the mass peak for the extension product completely disappeared, whereas the single peak corresponding to the cleavage product appeared at 5704 m/z (6180 − 476) indicating efficient cleavage.
To optimize the desalting process, various concentrations of ssDNA (4207: 5′-CTCTCTGGCCGCGT-3′) in 5 μL of 100 mM TCEP (pH 9.0) were introduced into the desalting channel at different flow rates, followed by a DI water wash at a flow rate of 20 μL min−1 for 10 min. Waste was collected, vacuum dried and dissolved in 5 μL of DI water. The concentrations of both input and output ssDNA solutions were measured with a NanoDrop instrument based on UV absorption using 100 mM TCEP (pH 9.0) as the reference. Approximately 20 pmol, 45 pmol and 100 pmol of ssDNA were introduced into the desalting channel at 1 μL min−1, which was then washed with DI water. After desalting, the total amounts of ssDNA not adsorbed by the C18 resin were about 1 pmole, 5 pmol and 40 pmol (Fig. 6A). These results indicate that the binding capacity of the C18 microspheres packed in the desalting channel is approximately 55 pmol under a constant flow rate of 1 μL min−1. This amount of DNA is far higher than the detection limit of MS.28 Theoretically, the binding capacity and recovery efficiency increases with a larger binding interface at a constant flow rate, which could be achieved by a larger number of C18 particles in the fixed stationary phase.29 However, this would build up back pressure due to the requirement for a longer microfluidic channel.30 On the other hand, the size of C18 microspheres is inversely proportional to the binding capacity and recovery efficiency.27 The use of larger particles would result in a lower surface-to-volume ratio due to the geometrical restriction of the microchannel. But, smaller particles also significantly contribute to the accumulation of back pressure.30 Therefore, the trade-off between channel length/particle size and binding capacity/recovery efficiency is an important design consideration.
In addition, we evaluated the effect of flow rate on desalting efficiency. Approximately 45 pmol of ssDNA in 5 μL of 100 mM TCEP (pH 9.0) was flowed into the desalting channel at 0.2 μL min−1, 1 μL min−1, or 5 μL min−1, followed by DI water wash. After desalting, the total amounts of ssDNA not adsorbed by the C18 beads were 1 pmole, 5 pmol and 14 pmol (Fig. 6B) respectively. These results imply that the binding capacity and recovery efficiency of the C18 microspheres packed in the desalting channel decreases as the flow rate increases, since the interaction time between the analyte and stationary phase (microspheres) is reduced.29 However, lower flow rates result in longer operation time, significantly decreasing the assay efficiency. Therefore, the optimal selection of flow rate is another essential design consideration.
mp = mr + mn − mb − mc | (1) |
To validate the multiplex genotyping capability, we used a synthetic template with 4 known positions as the query loci to mimic multiplex SNP genotyping. The SBE reaction contained 40 pmol each of the 4 SBE primers in a total volume of 10 μL. The final product masses can be calculated according to eqn (1). The primer sequences, molecular weights of primer and their potential final genotyping products are listed in Table 1, while their annealing sites are shown in Fig. 7C. As shown in Fig. 7B, nucleotides at these four sites were unambiguously identified. These results demonstrate the feasibility of using this microfluidic device for integrated and miniaturized SNP multiplex genotyping with high accuracy and sensitivity, having the potential for high-throughput and fully automated nucleotide variant detection. In addition, compared with SBE based genotyping on conventional platforms, the device employs on-chip temperature control, which allows for efficient temperature equilibration in the microchamber, significantly shortening thermal cycling times during amplification. This capability, as well as the ability to accommodate DNA fragments of multiple sizes, also provides the promise for microfluidic mini-MS sequencing, in which the fragments generated from Sanger dideoxyribonucleotide reactions are identified via MS at single nucleotide resolution, rather than by traditional gel electrophoresis and fluorescence detection.24 With the accuracy and sensitivity of MS, the device has great potential for accurate sequencing of small but highly polymorphic regions.24
Query Site (“SNPs”) | Primer Sequence (5′–3′) | Primer mass (Da) | Mass of final genotyping product (Da) | |||
---|---|---|---|---|---|---|
ddATP-N3-biotin | ddGTP-N3-biotin | ddCTP-N3-biotin | ddUTP-N3-biotin | |||
a Note: the masses of genotyping products shown in bold and italic type refer to the expected extension products using the above template shown in Fig. 7. | ||||||
a | TCTCTGGCCGCGTGTCT | 5144 | 5594 | 5723 | 5571 | 5685 |
b | CTCTCTGGCCGCGT | 4207 | 4657 | 4786 | 4634 | 4748 |
c | TAGATGACCCTGCCTTGTCG | 6084 | 6534 | 6663 | 6511 | 6625 |
d | GATAGGACTCATCACCA | 5163 | 5613 | 5742 | 5590 | 5704 |
The device consists of two microchambers situated on a temperature control chip for respectively carrying out SBE and SPP reactions, as well as a microchannel for desalting. These functional components are connected in series by microchannels with semi-circular cross sections and can be controlled by microfabricated elastomeric valves actuated by the underlying oil-filled channels driven by pressure. Moreover, integrated temperature sensors and heaters beneath the SBE and SPP microchambers allow independent, closed-loop control of chamber temperatures. The surfaces of the SBE microchamber are coated with Parylene C to prevent sample loss during thermal cycling. Streptavidin coated microbeads and C18 bonded silica microspheres are fully packed in the SPP chamber and desalting channel respectively to extract SBE products and remove salt in preparation for MS. Due to the serial design, array-based high throughput integration can be simply achieved by parallel control of the above described individual functional units using microvalves. Hence, each of the units can carry out multiplex SNP genotyping of a single sample, and multiple samples can be processed in parallel using the array-based configuration.
The temperature control units were characterized experimentally to ensure localized and efficient heating, as well as temporal accuracy of the temperature field. We demonstrated 100% extension of primer with cleavable mass-tagged ddNTPs and efficient chemical release of purified SBE products, with shortened temperature cycling time due to higher heat transfer efficiency of on-chip temperature control, leading to improved single-base resolution and ease of product extraction. The device's capability of SPP and desalting was tested at different flow rates. The efficiency of both SPP and desalting were shown to be flow rate dependent. Although lower flow rate leads to higher capture efficiency, it also dramatically increases the total operation time for the microfluidic genotyping. Therefore, the tradeoff between the efficiency of SPP/desalting and that of the full assay is an essential design consideration. Finally, uniplex and 4-plex assays on a mock synthetic template, which mimic the SNP detection process, were carried out within the fully integrated device. The accurate detection of these “SNPs” demonstrates the feasibility of using the microfluidic device for rapid, automated, integrated and miniaturized multiplex SNP genotyping with high accuracy and sensitivity. Potentially, it can also be used for high-throughput and fully automated nucleotide variant detection and accurate indel mini-sequencing. In addition, due to the serial design, arrays of this microfluidic device can be simply achieved on single chips by concerted control of the individual functional units arrayed in parallel to allow analysis of samples from many different patients. Such array-based devices can be created essentially by the same process used to fabricate a single device unit.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra44091e |
‡ These authors contributed equally to this paper. |
This journal is © The Royal Society of Chemistry 2014 |