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Sensitive Detection of microRNA with Isothermal Amplification and a Single-Quantum-Dot-Based Nanosensor Yan Zhang and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China ABSTRACT: MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are associated with various diseases. Here we develop a rapid, highly sensitive, and specific miRNA assay based on the two-stage exponential amplification reaction (EXPAR) and a single-quantum-dot (QD)-based nanosensor. The two-stage EXPAR involves two templates and two-stage amplification reactions under isothermal conditions. The first template enables the amplification of miRNA, and the second template enables the conversion of miRNA to the reporter oligonucleotide. Importantly, different miRNAs can be converted to the same reporter oligonucleotides, which can hybridize with the same set of capture and reporter probes to form sandwich hybrids. These sandwich hybrids can be assembled on the surface of 605 nm emission QDs (605QDs) to form the 605QD/reporter oligonucleotide/Cy5 complexes, where the 605QD functions as both a fluorescence resonance energy transfer donor and a target concentrator. Upon excitation with a wavelength of 488 nm, distinct Cy5 signals can be observed in the presence of target miRNA. This assay is highly sensitive and specific with a detection limit of 0.1 aM and can even discriminate single-nucleotide differences between miRNA family members. Moreover, in combination with the specific templates, this method can be applied for multiplex miRNA assay by simply using the same set of capture and reporter probes. This highly sensitive and specific assay has potential to become a promising miRNA quantification method in biomedical research and clinical diagnosis.
M
icroRNAs (miRNAs) are small, endogenous noncoding RNAs with approximately 22 nucleotides that are processed from large hairpin precursors.1 The miRNAs can suppress gene expression through incorporation into an active RNAinduced silencing complex (RISC).1,2 Recently, much evidence indicates that miRNAs play important roles in a variety of biological processes such as cell differentiation, apoptosis, proliferation, and immunological response.1,3,4 Especially, aberrant expression of miRNAs is associated with cancer initiation, tumor stage, and tumor response to treatments.5 Recent research also demonstrates that miRNAs can be used as biomarkers for diagnosis and prognosis and as targets for the discovery of new drugs.6,7 Consequently, the development of rapid and sensitive methods for identification and quantification of miRNAs is highly desirable in biomedical research and clinical diagnosis. To date, numerous analytical approaches have been developed for miRNA detection.8 Among them, Northern blotting,9 microarrays,10 and real-time quantitative polymerase chain reaction (qRT-PCR)6,11 are considered as the standard methods. However, Northern blotting is time-consuming with poor sensitivity and low throughput and usually requires large amounts of samples, which limits its application in clinical diagnosis. For microarray assay, the small size of miRNA, different melting temperatures, and high sequence homology between miRNA family members present major challenges to the sample labeling and r 2011 American Chemical Society
the probe design. In addition, microarray suffers from crosshybridization and poor laboratory-to-laboratory reproducibility.12 qRT-PCR has the advantages of practical ease, high sensitivity, and accuracy,6,11 but the short length of miRNA sophisticates the design of the primers, e.g., stemloop primer and locked nucleic acid (LNA)-modified primer, increasing the experimental cost and complexity. In addition, qRT-PCR requires the precise control of temperature cycling for successful amplification. To improve the detection sensitivity, flexibility, and adaptability, various new strategies have been developed, such as gold nanoparticle-based colorimetric assay,13 fluorescence-based assay,14 bioluminescence-based assay,15 enzymatic assay,1618 bead-based assay,19 and deep-sequencing-based assay.20 The gold nanoparticlebased colorimetric assay13 is simple and relatively inexpensive, but it suffers from nonspecific binding. For the fluorescence-based assay,14 each miRNA requires a specific probe, which increases the cost of multiplex detection. The bioluminescence-based assay makes use of the bioluminescent protein of Renilla luciferase (Rluc) as the label15 and is very simple and rapid, but its drawback is signal-off instead of signal-on. Enzymatic methods such as the horseradish peroxidase (HRP)-linked oligonucleotide assay,16 Received: September 10, 2011 Accepted: November 21, 2011 Published: November 21, 2011 224
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Table 1. Sequences of the Oligonucleotides sequence (50 30 ) reporter probe
Cy5-GTT ACC TTG ACT AGC
capture probe
TAC GAT AAG ACA GAG-biotin
reporter oligonucleotide Y
TCT GTC TTA TCG TAG CTA GTC AAG GTA A
X0 X0 template (let-7a)
AAC TAT ACA ACC TAC TAC CTC AAA CAG ACT CAA ACT ATA CAA CCT ACT ACC TCA A-P
X0 Y0 template (let-7a)
TTA CCT TGA CTA GCT ACG ATA AGA CAG ATC CAG ACT CAA ACT ATA CAA CCT ACT ACC TCA A-P
X0 X0 template (miR-21)
TAG CTT ATC AGA CTG ATG TTG AAA CAG ACT CAT AGC TTA TCA GAC TGA TGT TGA A-P
X0 Y0 template (miR-21)
TTA CCT TGA CTA GCT ACG ATA AGA CAG ATC CAG ACT CAT AGC TTA TCA GAC TGA TGT TGA A-P
let-7a let-7b
UGA GGU AGU AGG UUG UAU AGU U UGA GGU AGU AGG UUG UGU GGU U
let-7c
UGA GGU AGU AGG UUG UAU GGU U
miR-21
UCA ACA UCA GUC UGA UAA GCU A
invader assay,17 and use of signal-amplifying ribozymes18 provide immense signal amplification, but they suffer from poor specificity and low sensitivity. The bead-based assay has the advantages of high accuracy and low cost and is useful for high-throughput miRNA profiling, but its procedure is relatively labor-intensive.19 The deep-sequencing-based assay might be used for rapid evaluation of absolute miRNA levels, but it is usually costly and less accurate due to the introduction of some errors in several steps.20 Therefore, it is imperative to develop novel approaches with ultrasensitivity and high specificity for miRNA detection. Recently, the isothermal exponential amplification reaction (EXPAR) as an alternative amplification technique has been used for DNA and miRNA detection.2124 In comparison with conventional PCR with the involvement of thermal cycling, EXPAR proceeds at a constant temperature.2124 Moreover, EXPAR provides high amplification efficiency, which can rapidly amplify short oligonucleotides (106109-fold) within minutes.2124 In the EXPAR assay, SYBR Green I is usually used as the label, but the preferential binding of SYBR Green I to GC-rich sequences limits the analysis of multiple amplicons in multiplex real-time PCR.25 In addition, SYBR Green I shows both dose-dependent inhibition of the PCR reaction and promotion of nonspecific amplification.26 To overcome these problems associated with SYBR Green I, we turn to novel semiconductor nanocrystals— quantum dots (QDs). QDs have significant advantages over organic fluorophores, such as broad excitation, size-tunable photoluminescence spectra with narrow emission bandwidth, exceptional photochemical stability, and relatively high quantum yield.27 QDs have been widely used as fluorescent markers in genomic analysis, immunoassay, and in vitro and in vivo imaging.28,29 Recently, QDs have also been used as fluorescence resonance energy transfer (FRET) donors in a variety of biosensors to detect low-abundance nucleic acids, proteins, cocaine, and RNApeptide interaction.3034 In comparison with the conventional ensemble fluorescence measurements, the combination of QDs with single-molecule detection35,36 has shown significant advantages of high signal-to-noise ratio and improved sensitivity.37,38 More importantly, a single-QD-based nanosensor performs a homogeneous assay without the need for separating the binding probes from the free ones and can even eliminate the interference from the background fluorescence.38 Herein, we develop for the first time a novel miRNA detection method based on the two-stage EXPAR and a single-QD-based nanosensor. Isothermal amplification and the single-QD-based nanosensor offer improved sensitivity and selectivity for miRNA assay.
This method is capable of achieving a detection limit of as low as 0.1 aM and can even discriminate single-nucleotide differences between miRNA family members.
’ EXPERIMENTAL SECTION Sample Preparation. All HPLC-purified DNA oligonucleotides, miRNA, diethylpyrocarbonate (DEPC)-treated water, and RNase inhibitor were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The sequences of the oligonucleotides are listed in Table 1. Nicking endonuclease Nt.BstNBI and the Vent (exo) DNA polymerase were obtained from New England Biolabs (Beverly, MA). SYBR Green I (20 stock solution in dimethyl sulfoxide, 20 μg mL1) was purchased from Xiamen Bio-Vision Biotechnology (Xiamen, China). Streptavidin-coated 605 nm emission QDs (605QDs) were obtained from Quantum Dot Corp. (Hayward, CA). First-Stage EXPAR Reaction. The reaction mixtures for the first-stage EXPAR amplification were prepared separately as part A and part B. Part A consisted of 100 nM X0 X0 template, 250 μM deoxynucleotide triphosphates (dNTPs), 0.8 U μL1 RNase inhibitor, 0.5 Nt.BstNBI buffer (25 mM TrisHCl, pH 7.9, 50 mM NaCl, 5 mM MgCl2, 0.5 mM dithiothreitol), and miRNA. Part B consisted of 0.4 U μL1 Nt.BstNBI nicking enzyme, 0.05 U μL1 Vent (exo) DNA polymerase, 0.4 μg mL1 SYBR Green I, 1 ThermoPol buffer (20 mM TrisHCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-10), and DEPC-treated water. These two parts were prepared at 4 °C and mixed immediately before being placed in the real-time PCR system. The first-stage EXPAR reaction was performed in a volume of 10 μL at 55 °C, and the fluorescence intensity was monitored at intervals of 30 s with a LightCycler 480 real-time PCR system (Rotkreuz, Switzerland). Two-Stage EXPAR Reaction. To optimize the molar ratio of two templates, various ratios of X0 X0 template to X0 Y0 template (the total concentration of two templates was 500 nM) were employed in the two-stage EXPAR in the presence of 1 pM let-7a miRNA. The products of amplified reaction were analyzed by nondenaturating polyacrylamide gel electrophoresis (PAGE). The reaction mixtures for two-stage EXPAR amplification were prepared separately as part A and part B. Part A contained X0 X0 template, 375 μM dNTP, 0.5 Nt.BstNBI buffer, miRNA, and DEPC-treated water. Part B contained 0.8 U μL1 Nt.BstNBI nicking enzyme, 0.1 U μL1 Vent (exo) DNA polymerase, 1 ThermoPol buffer, 0.8 U μL1 RNase inhibitor, and X0 Y0 template. The reactions were carried out in a volume of 20 μL. 225
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Scheme 1. Scheme of the miRNA Assay Based on the Two-Stage EXPAR and Single-QD-Based Nanosensora
Key: (a, b) Exponential amplification of miRNA through the first-stage EXPAR reaction, (c) conversion of miRNA to the reporter oligonucleotide Y through the second-stage EXPAR reaction, (d) hybridization of the reporter oligonucleotide Y with two probes, (e) formation of the 605QD/reporter oligonucleotide Y/Cy5 complex through streptavidinbiotin binding, (f) fluorescence emission from Cy5 as a result of FRET between 605QD and Cy5 at an excitation wavelength of 488 nm. a
Part A was heated at 95 °C for 3 min to denature the X0 X0 template and miRNA. After part A was cooled to 55 °C, part B was added. Then the reactions were processed at 55 °C for 20 min and stored at 4 °C until analysis with nondenaturing polyacrylamide gel. A 14% polyacrylamide gel was prerun at room temperature in 0.5 Tris/borate/EDTA (TBE) buffer at 110 V for 30 min. Then 20 μL amplification products were mixed with 4 μL of 6 loading buffer and loaded onto the prerun gel to run at 25 °C for 50 min. The gel was stained with ethidium bromide and imaged by an ultraviolet (UV) transilluminator and a charge-coupled device (CCD) camera. Band intensities in the gels were analyzed by Quantity One software. To optimize the amplification time, EXPAR was performed at a molar ratio of the X0 X0 template of 0.4 in the presence of 1 pM let-7a miRNA for 10, 20, and 30 min, respectively. The amplification products were analyzed with the same method described above. Hybridization Reaction. The hybridization reaction was carried out in a buffered solution containing 100 mM TrisHCl, 10 mM (NH4)2SO4, and 3 mM MgCl2, pH 8.0. The Cy5-labeled reporter probes and biotinylated capture probes and their complementary oligonucleotides of reporter Y were incubated at 42 °C for 30 min to form sandwich hybrids (the molar ratio of the two labeled probes was kept at 1:1). After the sandwich hybrids were cooled to room temperature, streptavidin-coated 605QDs were added
to capture them to form the 605QD/reporter oligonucleotide Y/Cy5 complexes. Steady-State Fluorescence Measurements. All fluorescence spectra were measured at 20 °C on a fluorescence spectrometer (FSP920, Edinburgh Instruments, Livingston, U.K.). Emission spectra were recorded over the wavelength range of 550750 nm at a scan rate of 2 nm/s with an excitation wavelength of 488 nm. An excitation and emission slit width of 2.5 nm was used. Experimental Setup for Single-Molecule Detection. A 488 nm argon laser was used as the excitation light source. The 488 nm beam was reflected by a dichroic mirror (Z488RDC, Chroma Technology Corp., Rockingham, VT) and then focused by an oil immersion 100/1.30 numerical aperture (NA) objective lens (Olympus America, Inc., Melville, NY) to excite a sample in a 50 μm i.d. capillary. The sample passed a laser-focused detection volume through pressure-driven flow with a syringe pump (Harvard Apparatus, Holliston, MA). The flow rate in all measurements was kept at 1 μL min1. Photons emitted from 605QD and Cy5 were collected through the same objective, passed through the first dichroic mirror followed by a 50 μm pinhole (Melles Griot Co., Irvine, CA), and then separated by a second dichroic mirror (645 DCLP, Chroma Technology). After separation, photons emitted from Cy5 were filtered by a bandpass filter (D680/30M, Chroma Technology) and detected by an 226
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avalanche photodiode (model SPCM-AQR-13, EG&G, Vaudreuil, PQ , Canada) in the acceptor channel. At the same time, photons emitted from 605QD were filtered by a band-pass filter (D605/ 20M, Chroma Technology) and detected by an avalanche photodiode in the donor channel. A program written with Labview (National Instruments, Austin, TX) and a digital counter (National Instruments) were used to perform data acquisition and online data analysis. Fluorescence signals from both donor and acceptor channels were integrated in a 1 ms interval for a total running time of 100 s for each experiment. In single-molecule detection, a threshold was used to distinguish the single-molecule fluorescence signal from random fluctuation in the background. The threshold value was determined by evaluating data from the control sample. In this study, a threshold of 15 photon counts ms1 was set for Cy5 and a threshold of 10 photon counts ms1 was set for the 605QD. A burst was defined as a peak in a filtered data stream that exceeded a preset threshold. Burst counts are defined as the number of bursts detected within a certain running time.38
’ RESULTS AND DISCUSSION Principle of miRNA Assay. The principle of miRNA assay is shown in Scheme 1. The two-stage EXPAR involved two templates and two-stage amplification reactions under isothermal conditions.22 The first-stage reaction (Scheme 1a,b) was an exponential amplification with the involvement of the X0 X0 template, which enabled the amplification of miRNA. The second-stage reaction (Scheme 1c) was a linear amplification with the involvement of the X0 Y0 template, which enabled the conversion of miRNA to the reporter oligonucleotide Y. The details of the two-stage EXPAR reaction were as follows: The X0 X0 template contained two repeat sequences of X0 which were complementary to the target miRNA. The EXPAR reaction was initiated through the hybridization of the X0 X0 template with the target miRNA (Scheme 1a). The formation of a partial duplex can be extended by Vent (exo) DNA polymerase, resulting in a stable double-stranded DNA duplex with a recognition site for the nicking enzyme Nt.BstNBI. The cleavage of the upper DNA strand by the nicking enzyme Nt.BstNBI generated a short single-stranded DNA, X0. The sequence of X0 was the same as that of the target miRNA except for the change of U to T and the change of ribonucleotides to deoxyribonucleotides. X0 can act as a new primer to prime other X0 X0 templates, leading to an exponential amplification (Scheme 1b). The newly formed X0 can also serve as a primer to bind the second template, X0 Y0 , and produce the reporter oligonucleotide Y through a polymerization, nicking, and displacement reaction cycle. Unlike the X0 X0 template, the X0 Y0 template contained two different sequences of X0 and Y0 . The newly formed reporter oligonucleotide Y cannot in turn prime the X0 Y0 template, thus resulting in a linear amplification (Scheme 1c). It should be noted that different miRNAs can be converted to the same reporter oligonucleotides, which can be detected with the same set of capture and reporter probes without the need for resynthesis of the specific DNA probes for each new target miRNA or reoptimization of the assay conditions. After amplification, the reporter oligonucleotide Y was sandwiched by a biotinylated capture probe and a Cy5-labeled reporter probe (Scheme 1d). This sandwich hybrid was then assembled on the surface of a 605QD to form the 605QD/ reporter oligonucleotide Y/Cy5 complex through specific biotin streptavidin binding (Scheme 1e). When this complex was
Figure 1. (A) Real-time fluorescence monitoring of the first-stage amplification reaction triggered by different concentrations of let-7a miRNA. (B) Variance of the POI value as a function of the concentration of let-7a miRNA. Error bars show the standard deviation of three experiments.
excited by a 488 nm argon laser, the fluorescence signals of 605QD and Cy5 were observed simultaneously due to FRET from 605QD to Cy5 (Scheme 1f). The reasons for the selection of 605QD and Cy5 as an energy-transfer pair in this nanosensor were as follows: (1) no cross-talk between the emission spectrum of 605QD and that of Cy5, (2) no direct excitation of Cy5 at an excitation wavelength of 488 nm, (3) a high quantum yield (∼0.6) for 605QD and a high extinction coefficient (∼250 000 M1cm1) for Cy5, (4) the capability of a single 605QD to couple multiple reporter oligonucleotide Y/Cy5 complexes, which resulted in improved FRET efficiency.38,39 Real-Time Fluorescence Monitoring of First-Stage Amplification. To evaluate the amplification of miRNA in the firststage EXPAR reaction, the fluorescence in response to different concentrations of let-7a miRNA was monitored in real time by using SYBR Green I as the label.2124 When the amplification converted the single-stranded X0 X0 template to a partially double-stranded DNA duplex, a sigmoidal curve was obtained (Figure 1A). The point of inflection (POI), which is defined as the time corresponding to the maximum slope in the sigmoidal curve, was used for the quantitative detection of let-7a miRNA. As shown in Figure 1B, a linear correlation was obtained between the POI values and the logarithm (log) of the miRNA concentration in the ranges of 0.1 μM to 0.1 nM and 0.1 nM to 0.1 fM, respectively. The correlation equations are POI = 1391.39 258.61 log10 C (the correlation coefficient is 0.997) and POI = 665.86 53.07 log10 C (the correlation coefficient is 0.993), respectively; C is the concentration of let-7a miRNA. 227
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These results indicated that the first-stage amplification proceeded exponentially and reached the maximum plateau rapidly. Optimization of the Two-stage Amplification Reaction. In the two-stage EXPAR reaction, the first template, X0 X0 , enabled amplification of miRNA and the second template, X0 Y0 , converted miRNA to the reporter oligonucleotide Y. Since the total concentration of two templates was kept constant in the experiments, the yield of reporter oligonucleotide Y was particularly sensitive to the ratio of two templates.22 In addition, because both of the templates contained the same sequence X0 , they might competitively hybridize with miRNA, consequently influencing the final production of reporter oligonucleotide Y. Therefore, it was necessary to optimize the molar ratio of the two templates. The production of reporter oligonucleotide Y was analyzed by nondenaturating PAGE (Figure 2A). To quantitatively evaluate the influence of the molar ratio of the X0 X0 template, the level of reporter oligonucleotide Y production was calculated on the basis of following equation: level of reporter Y production ¼
Y N 100% Y
where Y is band intensity value of reporter oligonucleotide Y in the presence of miRNA and N is the band intensity value of the negative control without miRNA. As shown in Figure 2A, at a molar ratio of the X0 X0 template of 0.4, a well-defined band of reporter oligonucleotide Y (28 nt) was observed (lane 3 in Figure 2A) in the presence of let-7a miRNA and the level of reporter oligonucleotide Y production was calculated to be 91.8% (Figure 2B). In contrast, the negative control without let-7a miRNA showed a negligible band (lane 7 in Figure 2A). When the molar ratio of the X0 X0 template was 0.2, 0.6, and 0.8, the pixel intensity of observed bands decreased even in the presence of let-7a miRNA (lanes 2, 4, and 5 in Figure 2A) as compared to that at a molar ratio of 0.4 (lane 3 in Figure 2A), whereas the pixel intensity of the bands from their negative control kept a high level (lanes 6, 8, and 9 in Figure 2A). The level of reporter oligonucleotide Y production was calculated to be 66.1%, 54.3%, and 44.4% when the molar ratio of the X0 X 0 template was 0.2, 0.6, and 0.8, respectively (Figure 2B), much less than that at a molar ratio of 0.4. Therefore, the optimum molar ratio of the X0 X0 template was selected to be 0.4 in the subsequent experiments. The low yield of reporter oligonucleotide Y at lower molar ratio of the X0 X0 template (0.2) might be attributed to the following two factors: (1) The newly formed miRNA in the first-stage reaction was sequestered by the high-concentration X0 Y0 template, which hindered the amplification of miRNA and subsequently suppressed the second-stage reaction. (2) The highconcentration single-stranded X0 Y0 template might be easily bound by DNA polymerase and synthesized in an unprimed fashion, increasing the nonspecific background amplification.40 The low yield of reporter oligonucleotide Y at higher molar ratio of the X0 X0 template (0.6 and 0.8) might be attributed to the following two factors: (1) The high-concentration X0 X0 template enabled efficient amplification of miRNA in the first-stage reaction, but the low-concentration X0 Y0 template cannot effectively convert the miRNA to the reporter oligonucleotide Y in the second-stage reaction. (2) The interaction between the high-concentration X0 X0 template and the DNA polymerase made contributions to the nonspecific background amplification as well.40 The influence of the amplification time on the reporter oligonucleotide Y production was further investigated. As shown
Figure 2. Optimization of the two-stage amplification reaction. (A) Effect of the molar ratio of the X0 X0 template on the reporter oligonucleotide Y production in the presence of let-7a (lanes 2, 3, 4, and 5) and in the absence of let-7a (lanes 6, 7, 8, and 9). Key: lane M, DNA ladder marker; lane 1, reporter oligonucleotide Y (28 nt); lanes 2 and 6, molar ratio of the X0 X0 template 0.2; lanes 3 and 7, molar ratio of the X0 X0 template 0.4; lanes 4 and 8, molar ratio of the X0 X0 template 0.6; lanes 5 and 9, molar ratio of the X0 X0 template 0.8. (B) Variance of the level of reporter oligonucleotide Y production as a function of the molar ratio of the X0 X0 template. (C) Variance of the level of reporter oligonucleotide Y production as a function of the amplification time. The concentration of let-7a miRNA was 1 pM, and the total concentration of the X0 X0 template and X0 Y0 template was 500 nM. Error bars show the standard deviation of three experiments. 228
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Figure 5. Variance of Cy5 burst counts as a function of the miRNA concentration. The concentration of 605QD was 2 1011 M, the concentration of the Cy5-labeled reporter probe was 4.8 1010 M, and the concentration of the biotinylated capture probe was 4.8 1010 M. Error bars show the standard deviation of three experiments.
Figure 3. Variance of 605QD and Cy5 fluorescence as a function of the ratio of the reporter oligonucleotide Y/Cy5 complex to 605QD in the 605QD/reporter oligonucleotide Y/Cy5 complex. The concentration of 605QD was 1 108 M, and the concentration of the reporter oligonucleotide Y/Cy5 complex was varied with the ratio of the reporter oligonucleotide Y/Cy5 complex to 605QD as shown.
the maximum when the amplification time was 20 min. Therefore, the optimum amplification time was selected to be 20 min in the subsequent experiments. Fluorescence Measurement of QD-Based FRET. To evaluate the influence of the Cy5-to-605QD ratio on the FRET efficiency, we measured the 605QD and Cy5 fluorescence at various ratios of reporter oligonucleotide Y/Cy5 complexes to 605QD. As shown in Figure 3, when the ratio of reporter oligonucleotide Y/Cy5 complexes to 605QD increased, the 605QD fluorescence decreased while the Cy5 fluorescence increased correspondingly, suggesting FRET between 605QD and Cy5 in the 605QD/reporter oligonucleotide Y/Cy5 complex. Assuming that each 605QD was conjugated with 1215 streptavidins and each streptavidin had 3 available biotin-binding sites after conjugation to the 605QD, in principle, up to ∼3645 reporter oligonucleotide Y/Cy5 complexes can be captured by a single 605QD.33,38,41 As a result, the Cy5 fluorescence signal was significantly amplified through enhanced energy transfer between 605QD and Cy5 by increasing the number of Cy5 acceptors linked to a 605QD donor.33,38,41 To ensure high detection sensitivity, a Cy5-to-605QD ratio of 24:1 was used in the following experiments. The single-QD-based nanosensor was further applied to detect the reporter oligonucleotide Y at the optimum Cy5-to605QD ratio of 24:1. In the presence of reporter oligonucleotide Y, the fluorescence signals of 605QD and Cy5 were observed simultaneously owing to FRET between the 605QD and Cy5 in the 605QD/reporter oligonucleotide Y/Cy5 complex (Figure 4A). In contrast, in the control group without reporter oligonucleotide Y, only the 605QD signals were observed in the donor channel, but no Cy5 signal was observed in the acceptor channel (Figure 4B). Since this assay was carried out in a microfluidic flow at the singleparticle level, the free Cy5-labeled reporter probes were far away from the 605QD in the absence of reporter oligonucleotide Y. Consequently, neither false FRET nor Cy5 fluorescence signals were observed in the control group (Figure 4B). This near-zero background noise observed in the negative control was crucial for the high sensitivity of miRNA assay. Sensitivity of miRNA Assay. We further employed the singleQD-based nanosensor in combination with the two-stage EXPAR to detect different concentrations of let-7a miRNA. Under the optimal conditions as described above, let-7a miRNA was
Figure 4. (A) Representative traces of fluorescence bursts from Cy5 and 605QD in the presence of reporter oligonucleotide Y. (B) Representative traces of fluorescence bursts from Cy5 and 605QD in the control group without reporter oligonucleotide Y. The concentration of 605QD was 2 1011 M, the concentration of Cy5-labeled reporter probe was 4.8 1010 M, and the concentration of the biotinylated capture probe was 4.8 1010 M.
in Figure 2C, at the optimum molar ratio of the X0 X0 template of 0.4, the level of reporter oligonucleotide Y production reached 229
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sensitivity might be attributed to the combination of high amplification efficiency of EXPAR,2124 high sensitivity of single-particle detection,33 and near-zero background noise of the single-QD-based nanosensor.38 let-7 miRNA family members regulated three major oncogenes, including RAS, MYC, and HMGA2.42 A very low expression level of let-7 miRNA could be correlated with advanced lung cancers.42 This ultrasensitive detection method might be further applied for accurate quantification of low-abundance miRNA in early clinical diagnosis. Specificity of miRNA Assay. Because of the high similarity of miRNA sequences, it was a great challenge to discriminate singlenucleotide differences between miRNA family members. To evaluate the specificity of the proposed miRNA assay, let-7 family members let-7a, let-7b, and let-7c miRNA were analyzed with the let-7a-specific templates. As shown in Table 1, there were two base mismatches near the 30 end of the miRNA between let-7a and let-7b. In the measurement of the singleQD-based nanosensor, the Cy5 burst counts from let-7a were approximately 42-fold more than those from let-7b (Figure 6A). Notably, the Cy5 burst counts from let-7a were approximately 8-fold more than those from let-7c (Figure 6A), even though there was only a single base mismatch near the 30 end of the miRNA between let-7a and let-7c (Table 1), suggesting the high specificity of this miRNA assay. In comparison with the previous methods for discriminating single-nucleotide differences between miRNA family members,14,43 this method had the significant advantage of high selectivity. For example, the reported single-molecule detection with two organic fluorophores as the labels gave signals of 3-fold difference between let-7a and let7c,14 while the ligase chain reaction (LCR)-based method gave a pixel intensity ratio of about 100:26 between let-7a and let-7c.43 This method might be further extended to multiplex miRNA assay. To demonstrate this concept, three samples with only let7a, only miR-21, and a mixture of let-7a and miR-21 were prepared. Each sample was equally divided into two aliquots, where either the let-7a-specific templates or the miR-21-specific templates (Table 1) were added. At last, with the addition of the same set of capture and reporter probes as well as streptavidincoated 605QDs, each aliquot was subjected to single-molecule detection. As shown in Figure 6B, in the sample with only let-7a, Cy5 signals were detected only in one aliquot with the addition of let-7a-specific templates, but no Cy5 signals were detected in the other aliquot with the addition of miR-21-specific templates. In the sample with only miR-21, Cy5 signals were detected only in one aliquot with the addition of miR-21-specific templates, but no Cy5 signals were detected in the other aliquot with the addition of let-7a-specific templates (Figure 6B). In the sample with the mixture of let-7a and miR-21, Cy5 signals were detected in both aliquots (Figure 6B). These results indicated that this method in combination with the specific templates can be applied for multiplex miRNA assay by simply using the same set of capture and reporter probes.
Figure 6. Specificity of miRNA assay. (A) Comparison of Cy 5 burst counts measured with the addition of let-7a-specific templates in the presence of let-7a, let-7b, and let-7c. The concentration of each let-7a, let-7b, and let-7c was 10 fM. (B) Multiplex miRNA assay. With the addition of the specific templates, let-7a and miR-21 were converted to the same reporter oligonucleotide Y, which could be detected with the same set of capture and reporter probes. The concentration of let-7a and miR-21 was 1 fM. Error bars show the standard deviation of three experiments.
converted to the reporter oligonucleotide Y through two-stage EXPAR. The reporter oligonucleotide Y then hybridized with the biotinylated capture probe and Cy5-labeled reporter probe to form the sandwich hybrid. These sandwich hybrids were caught and assembled on the surface of 605QDs through specific streptavidinbiotin binding and detected by the single-QDbased nanosensor at an excitation wavelength of 488 nm. As shown in Figure 5, the Cy5 burst counts increased as a function of increasing concentration of let-7a miRNA from 0.1 aM to 10 fM. Notably, the Cy5 burst counts in the presence of let-7a miRNA were significantly more than those of the negative control (p < 0.05) even when the concentration of let-7a miRNA was as low as 0.1 aM. As a result, the detection limit of the single-QD-based nanosensor might reach 0.1 aM, which is an improvement by as much as 4 orders of magnitude compared to that of previous research using the single-QD-based nanosensor but without the involvement of amplification.38 Moreover, in comparison with the reported EXPAR assay with SYBR Green I as the label,24 the sensitivity of the current assay improved by as much as 2 orders of magnitude. In addition, in comparison with the reported singlemolecule detection with two organic fluorophores as the labels,14 the sensitivity of the current assay improved by as much as 6 orders of magnitude. Such significant improvement in detection
’ CONCLUSIONS We have developed for the first time a rapid, sensitive, and specific miRNA assay based on the two-stage EXPAR and a singleQD-based nanosensor. The two-stage EXPAR offers distinct advantages of high amplification efficiency, isothermal nature, and rapid amplification kinetics. In addition, the single-QD-based nanosensor with near-zero background noise significantly improves 230
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Analytical Chemistry
ARTICLE
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’ ACKNOWLEDGMENT This work was supported by National Basic Research Program 973 (Grants 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grant 21075129), and the Knowledge Innovation Project of the Chinese Academy of Sciences (Grant KGCX2-YW-130). ’ REFERENCES (1) Bartel, D. P. Cell 2004, 116, 281–297. (2) Lee, Y.; Ahn, C.; Han, J. J.; Choi, H.; Kim, J.; Yim, J.; Lee, J.; Provost, P.; Radmark, O.; Kim, S.; Kim, V. N. Nature 2003, 425, 415–419. (3) Wienholds, E.; Plasterk, R. H. A. FEBS Lett. 2005, 579, 5911– 5922. (4) Kloosterman, W. P.; Plasterk, R. H. A. Dev. Cell 2006, 11, 441– 450. (5) Croce, C. M. Nat. Rev. Genet. 2009, 10, 704–714. (6) Raymond, C. K.; Roberts, B. S.; Garrett-Engele, P.; Lim, L. P.; Johnson, J. M. RNA 2005, 11, 1737–1744. (7) Bartels, C. L.; Tsongalis, G. J. Clin. Chem. 2009, 55, 623–631. (8) Cissell, K. A.; Deo, S. K. Anal. Bioanal. Chem. 2009, 394, 1109–1116. (9) Lagos-Quintana, M.; Rauhut, R.; Lendeckel, W.; Tuschl, T. Science 2001, 294, 853–858. (10) Thomson, J. M.; Parker, J.; Perou, C. M.; Hammond, S. M. Nat. Methods 2004, 1, 47–53. (11) Chen, C. F.; Ridzon, D. A.; Broomer, A. J.; Zhou, Z. H.; Lee, D. H.; Nguyen, J. T.; Barbisin, M.; Xu, N. L.; Mahuvakar, V. R.; Andersen, M. R.; Lao, K. Q.; Livak, K. J.; Guegler, K. J. Nucleic Acids Res. 2005, 33, e179. (12) Lagos-Quintana, M.; Rauhut, R.; Yalcin, A.; Meyer, J.; Lendeckel, W.; Tuschl, T. Curr. Biol. 2002, 12, 735–739. (13) Yang, W. J.; Li, X. B.; Li, Y. Y.; Zhao, L. F.; He, W. L.; Gao, Y. Q.; Wan, Y. J.; Xia, W.; Chen, T.; Zheng, H.; Li, M.; Xu, S. Q. Anal. Biochem. 2008, 376, 183–188. (14) Neely, L. A.; Patel, S.; Garver, J.; Gallo, M.; Hackett, M.; McLaughlin, S.; Nadel, M.; Harris, J.; Gullans, S.; Rooke, J. Nat. Methods 2006, 3, 41–46. (15) (a) Cissell, K. A.; Campbell, S.; Deo, S. K. Anal. Bioanal. Chem. 2008, 391, 2577–2581. (b) Cissell, K. A.; Rahimi, Y.; Shrestha, S.; Hunt, E. A.; Deo, S. K. Anal. Chem. 2008, 80, 2319–2325. (16) Su, X.; Teh, H. F.; Lieu, X. H.; Gao, Z. Q. Anal. Chem. 2007, 79, 7192–7197. (17) Allawi, H. T.; Dahlberg, J. E.; Olson, S.; Lund, E.; Olson, M.; Ma, W. P.; Takova, T.; Neri, B. P.; Lyamichev, V. I. RNA 2004, 10, 1153–1161. (18) Hartig, J. S.; Grune, I.; Najafi-Shoushtari, S. H.; Famulok, M. J. Am. Chem. Soc. 2004, 126, 722–723. 231
dx.doi.org/10.1021/ac202405q |Anal. Chem. 2012, 84, 224–231
