Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes
We report a novel microfluidic system that is capable of rapidly detecting DNA and its mutants in microfluidic droplets, in addition to elucidating the dynamic hybridization process. This microfluidic picoliter droplet analysis system is able to overcome the limitations of conventional analytical techniques that utilize immobilized sensing probes on a substrate. Molecular beacon (MB), a fluorescence resonance energy transfer (FRET) molecule, was used as the DNA sensing probe in picoliter droplets. The MB-DNA duplex formation process was analyzed by the change in FRET signal, which was acquired by the time-resolved method: converting distance traveled to hybridization time. This technique demonstrates the ability to detect presence of target nucleic acids within few seconds, multiplex DNA samples in microdroplet, and distinguish single nucleotide polymorphisms. It is promising for analyzing biomolecules or reactions, such as mRNA, cells, enzymatic activity, and protein folding whose analysis requires rapid mixing and small volume.
KeywordsMicrodroplet Molecular beacon FRET DNA analysis Time-resolved SNP
DNA hybridization is extensively applied in medicine for diagnosing genetic diseases and gene sequencing (Livak 1999; Syvanen 2001). Analysis of DNA sequences with single nucleotide polymorphism (SNP) is one of the major methods for diagnosing genetic diseases. Microarray(Lipshutz et al. 1999; van’t Veer et al. 2002), quartz crystal microbalance (QCM) (Caruso et al. 1997; Okahata et al. 1998) and surface plasmon resonance (SPR) (Thiel et al. 1997; He et al. 2000) are examples that have been developed to analyze DNA hybridization, sequencing and SNP. However, these methods are often carried out in bulk solutions with immobilized sensing probes on the substrate surface, which is dependent on the passive diffusion of DNA samples toward the sensing probes. The limited solid–liquid diffusion greatly hinders the hybridization rate of target DNA and sensing probe. Thus, the hybridization time for DNA and immobilized-probe normally takes from several hours to overnight (Peterson et al. 2001; Jobs et al. 2002). Additionally, conditions of sensing probes, such as the random immobilization density, electrostatic hindrance and the length of DNA probes on the substrate will all compromise the precision of dynamic readings (Steel et al. 2000; Peterson et al. 2001; Wong et al. 2005). Moreover, the stringent rinsing processes are also normally required after the hybridization process to remove non-specific binding. It further increases the whole process time. Therefore, it is important to develop a DNA analysis assay which is an easy, faster, accurate and low sample consumption method for multiplex DNA analysis.
Because of the small dimensions of the microfluidic system, the flows with low Reynolds number are always laminar. The diffusion distance was also greatly reduced in microfluidic channel. These unique intrinsic properties of microfluidic system contribute to the easily control of liquid behavior in microfluidic channel. Therefore, microfluidic based techniques were applied to overcome the slow hybridization problem caused by the diffusion-limited solid–liquid reaction, and further accelerate nucleic acid hybridization (Sanders and Manz 2000; Lagally et al. 2001; Sia and Whitesides 2003). Mixing in laminar flow system occurs by diffusion alone, which can result in unacceptable long mixing times (Kamholz et al. 1999; Liu et al. 2000). Moreover, uneven dispersion of DNA samples in solution and contamination on microfluidic channel surfaces can spoil accurate quantitative analysis. The DNA sample dispersion in continuous microfluidic system hinders the application of this technique for in situ multiplex DNA sample detection, and different concentrations of DNA sample analysis. Surface treatment is usually required to avoid DNA contaminants in the microfluidic channel.
The monodispersed picoliter microfluidic droplet generation system developed by our group and others (Tice et al. 2003; Tan et al. 2004; Hsieh et al. 2005; Teh et al. 2008) can serve as a promising micro-reactor for biological and chemical assays. It employs pressure-driven flow to inject aqueous solutions into aqueous immiscible solutions and form picoliter microdroplets. The biological or chemical reagents are all encapsulated in microdroplets and each droplet is isolated by the immiscible liquid (e.g. mineral oil), thus greatly reducing sample contamination on the microchannel side walls and eliminating reagent dispersion problems. The system is useful not only for DNA sample identification, but also for quantitative analysis. The liquid–liquid reaction rate of DNA hybridization in homogeneous liquid is about 40-fold faster than the hybridization rate in a solid–liquid interface (Gao et al. 2006). Hence, it is advantageous to improve the DNA sample/sensing probe hybridization rate using this technique rather than the conventional immobilized-probe approach. Compared to conventional microfluidics with continuous laminar flows, the microdroplet allows rapid mixing among reagents in droplet (Tice et al. 2003). The microdroplet formation system that we developed is capable of generating over five hundred monodisperse droplets per second with a size deviation of less than 2% (Tan et al. 2004). However, because of the fundamental differences between the microdroplet generation system and other conventional techniques, a liquid–liquid reaction based DNA sensing probe is needed to conduct DNA detection in microdroplets.
The distinctive biomolecular recognition and signal transduction capabilities of molecular beacons (MB) (Tyagi and Kramer 1996) have increasingly been applied to many medical diagnosis applications (Chen and Kwok 1997; Piatek et al. 1998) and biological assays, including genotyping for SNP (Kwok 2001; Syvanen 2001), multiplex genetic analysis (Marras et al. 1999; Vet et al. 1999), quantitative PCR (Vogelstein and Kinzler 1999; Chen et al. 2000), enzymatic reaction of proteins (Fang et al. 2000; Tung et al. 2000), and detection of mRNA in living cells (Sokol et al. 1998; Tsuji et al. 2000; Fang et al. 2002). MB is a class of fluorescence resonance energy transfer (FRET) molecules that are synthesized in a hairpin structure with stem and loop portions. The closed hairpin structure brings the donor fluorophor and quencher in close proximity to one another and thus quenches fluorescence. When the MB probes encounter complementary target DNA, the hairpin structure will open and the fluorescence is restored. The FRET mechanism is based on the non-radiative transfer of excitation from a donor fluorophor to an acceptor, and thus depends on the distance between the donor and acceptor, their relative orientation, and donor excited state lifetime. These outstanding properties enable FRET to be a sensitive label-free DNA detection probe in biological systems (Stryer and Haugland 1967; Dosremedios and Moens 1995; Deniz et al. 1999). The transition process between quenched and fluorescent states allows observation of bound and unbound target nucleic acids (Bonnet et al. 1999).
In order to achieve rapid DNA detection in homogeneous liquid phase with single nucleotide mismatch sensitivities, we used MB as the DNA sensing probe in the microdroplets. The target DNA and MB were all encapsulated in monodispersed picoliter droplet emulsions to reduce sample volume and to enhance the DNA detection efficiency. In this paper, we demonstrate a fast DNA sample and mutant detection in a few seconds, and evaluate dynamic MB-DNA duplex formation using label-free DNA analysis in microdroplet (LFDAM) system.
2 Materials and results
2.1 Wild and mutant DNA samples
The sequences of designed molecular beacon probes, target oligonucleotides, mutants, NS-DNA
As listed in Table 1, seven different sequences of oligonucleotides were synthesized as DNA samples for evaluating the selectivity and sensitivity of the designed molecular beacon probe. The SynBRCA1 is the complementary ssDNA (cDNA) for designed molecular beacon probe (MB-BRCA1). Strands with SNP, double nucleotide polymorphism’s (2NP), and triple nucleotide polymorphism’s (3NP), have been synthesized to understand how the number of mutations affects hybridization kinetics. To further evaluate the influence of the location of mutation points on the nucleic acid, two different SNPs and 2NPs were synthesized. SNP-M and SNP-E stand for the mutation at the middle and end of SynBRCA1 sequence respectively. The sequence of 2NP-M has both mutations at the middle, and the sequence of 2NP-ME has one point mutation in the middle and the other at the end of the SynBRCA1 sequence. The 3NP has all three mutations at the middle of the SynBRCA1 sequence. Non-specific ssDNA (NS-DNA), HepCV, is 25 nucleotides long, and has a portion of the wild type Hepatitis C virus gene sequence. All oligonucleotides were acquired from Integrated DNA Technologies, Inc (Coralville, IA).
2.2 Reagents and solutions
Mineral oil (Fisher Scientific, NH) used in this experiment contains 0.5% of Sorbitan Monooleate (Sigma-Aldrich, MO). Fluo-4 (Molecular Probes, CA) and CaCl2 (Fisher Scientific, NH) were used to verify the completion of the reagent mixing time point in the droplets. Fluo-4 is a Ca2+ indicator and emits fluorescence once it meets Ca2+ ions(Gee et al. 2000). Therefore, it is used to verify mixing completion inside droplets. All water used during the experiment was purified by NANOpure Water System (Thermo Fisher Scientific, MA) with a water resistance higher than 18.2 MΩ-cm and autoclaved. All buffer components were nuclease-free, and all dilutions and centrifuge microtubes were also purified by autoclaved, nuclease-free water.
2.3 Digital microfluidic device
The microfluidic channel was fabricated in polydimethylsiloxane (PDMS) elastomer (Sylgard 184 from Dow Corning, Midland, MI) using the soft-photolithography technique (Duffy et al. 1998). PDMS has excellent transparent optical properties and does not auto-fluoresce as most plastics do (Duffy et al. 1998), making it an ideal material for fluorescence analysis platforms involving biochemical reactions. The PDMS microchannel was bonded to a microscope cover slide after surface treatment of oxygen plasma. The reagents and samples were driven into the microchannel via polymer tubing (Tygon Microbore, OH) and nanoliter precision syringe pumps (Harvard Apparatus, MA).
We used a monochrome high-resolution deep cooled camera (Orcaerga, Hamamatsu USA, CA) as the fluorescence detector. Another monochrome high-speed camera system (Photron USA, CA), used for observing microdroplet generation and measuring droplet velocity, was also mounted to the Olympus inverted microscope system (IX51, Olympus USA, CA). The band pass filters sets, i.e. excitation filters, dichroic beam splitters, and long-pass emission filters, for both Cy3 and FAM fluorescence detection were all purchased from Chroma (Rockingham, VT). ImagePro Plus software (MediaCybernetics Inc., MD) was used for fluorescence data analysis.
3 Results and discussion
3.1 MB-DNA duplex formation evaluation
The DNA sample and MB-BRCA1 were injected into the microchannel, with concentrations of 2.5 and 1 μM respectively. Due to the monodispersity of microfluidic droplet generation, each droplet encapsulated the same number of MB and DNA. By accumulating fluorescence at one location, the fluorescence signal emitted from MB can be amplified using a longer exposure time without worrying about the change of different reaction stages. By recording fluorescence signal downstream of microchannel, MB-DNA duplex formation reaction can be observed at different stages. Droplets carrying MB-DNA duplex formation reaction stages are “captured by repeated images” and “frozen” along the microchannel.
The fluorescence restoration process of MB during the duplex formation with different cDNA or mutants in microdroplet is shown in Fig. 7. The hybridization signals at the channel turns were not collected. However, the 23 ms of time lapse in each turn was counted. The time axis covers from the mixing completion point to the end of hybridization region just before the outlet reservoir. The fluorescence reaches a plateau in Region 3. The fluorescence intensity from MB remains constant at this plateau region, representing the completion of MB-DNA duplex formation. Therefore, the LFDAM device used in this research can evaluate the dynamic hybridization kinetics in less than 10 s. Faster detection rates can be reached with higher DNA concentrations.
Photobleaching of fluorophors that are labeled on DNA has been an issue in many fluorescence analyses; it typically occurs in a matter of only few microseconds to seconds (Osborne et al. 2001; Yao et al. 2003). Specifically for Cy3, photobleaching manifests in a few seconds (Eggeling et al. 1998). In this experiment, the MB passes the 4× magnification observation window as quickly as 249 ms. The time-resolve fluorescence analysis method not only reduces the photobleaching problem while monitoring the real-time reaction, but it can also compensate for the short exposure of each droplet by accumulating repeating fluorescent microdroplets at the same location. In this time-resolved approach, the fidelity of fluorescence signals can be retained.
3.2 Dual DNAs detection in droplet
3.3 Dynamic range of linearity
The measured fluorescence intensity is directly proportional to sample concentration, droplet volume, droplet generation rate, and fluorescence acquisition time. The selection of donor fluorophor will affect the value of Io, and the selection of quencher will affect the value of Ic. The value of N is inversely proportional to the number of mutation points. In other words, these results disclose the characteristic probability of MB-BRCA1 closing and opening for different mutations of SynBRCA1.
We have developed a LFDAM system for using FRET molecules to rapidly detect DNA in Microdroplets and also capable evaluate the dynamic hybridization process through a time-resolved method. Because convection-driven mixing occurs in picoliter microdroplets, the often lengthy diffusion time in bulk solution or non-droplet microfluidic channels is greatly reduced. Consequently, the reoccurring identical droplets represent the hybridization stage via fluorescent signals, and the different reaction stages in droplets were shown at different traveling distances. The reaction time correlates to the distance traveled by the droplets in the microchannel. Since each droplet is exposed to excitation source for only 249 ms, the photobleaching problem was reduced to a minimum. The designed MB-BRCA1 for SynBRCA1 detection can reach a SNR as high as 128 demonstrating LFDAM as a sensitive platform to distinguish SNP point mutation at various sites and elicit the difference of their hybridization kinetics. Duplex target detection in one microdroplet was demonstrated by analyzing SynBRCA1 and HepCV in LFDAM. The limitation of detection in LFDAM with MB can reach as low as 500 fM. This microdroplet array and molecular beacon based system, LFDAM, is promising for high-throughput genotyping of SNPs in pharmacogenomics research, where the identification of variations in specific genes is necessary to evaluate drug efficacy, toxicity and metabolism. It will be useful for establishing optimal therapeutic strategies for individual patients. LFDAM may also be used to analyze other rapid biomolecular reactions, such as enzymatic activity, multiplex nucleic acid detection, cells, RNA, and proteins folding whose analyses require small volume, rapid mixing, and minimum effect of photobleaching.
The authors would like to gratefully acknowledge Yuh Adam Lin, Professor James Brody, Joseph Harris, Alan Lee, and Wajeeh Saadi of the Biomedical Engineering Department, University of California, Irvine for their precious suggestions and generosity in letting us use their lab equipments. This research was funded by Defense Advanced Research Projects Agency (DARPA) under the MF3 N/MEMS S&T Centers (HR001106-1-0050), and NASA through the University Affiliated Research Center (NAS2-03144).
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Hsieh AT, Hung LH, Lee AP (2005) Rapid breast cancer gene detection in picoliter droplet. The 9th international conference on miniaturized systems for chemistry and life sciences, BostonGoogle Scholar
- Tung CH, Mahmood U et al (2000) In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res 60(17):4953–4958Google Scholar
- van’t Veer LJ, Dai HY, et al (2002) Gene expression profiling predicts clinical outcome of breast cancer. Nature 415(6871):530–536Google Scholar
- von Ahsen N, Oellerich M et al (1999) Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: Prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler. Clin Chem 45(12):2094–2101Google Scholar