Sensitive Detection and Identification of DNA and RNA Using a

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Sensitive Detection and Identification of DNA and RNA Using a Patterned Capillary Tube Amos Bardea,† Noa Burshtein,‡ Yinon Rudich,‡ Tomer Salame,§ Carmit Ziv,§ Oded Yarden,§ and Ron Naaman*,† †

Department of Chemical Physics, The Weizmann Institute, Rehovot 76100, Israel Department of Environmental Sciences, The Weizmann Institute, Rehovot 76100, Israel § Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel ‡

bS Supporting Information ABSTRACT: We report on a new ultrasensitive and fast technique for the detection and identification of both DNA and RNA with sensitivity of a few molecules. The new method is based on a patterned capillary tube (PCT) in which the internal surface of a glass tube is patterned with rings of different single-stranded DNA probes. A solution containing single-stranded analyte flows through the tube. Upon hybridization of appropriate DNA and RNA from the solution, DNA polymerase and reverse transcriptase (RT) are employed to synthesize the complementary nucleic acids with deoxynucleoside triphosphate (dNTP) labeled with fluorophores. The sample-analyte hybrids are detected by their fluorescence signal. We show that the new method is sensitive, is specific, can detect simultaneously both DNA and RNA from the same sample, and allows detection of analytes in serum.

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he importance of sensitive DNA and RNA detection is increasing in several fields. In medicine, for example, it is essential for advancing the diagnostics and treatment of cancer, myocardial infarction, diseases caused by fungi, bacteria, and viruses1
3 and for the development of personalized medicine.4 DNA detection has proven to be a revolutionary tool in forensic sciences5,6 and in analyses of environmental samples.7 These various applications have stimulated the development of advanced methods for DNA and RNA detection and identification.8
10 They include optical-based methods like DNA chips11
13 and electrical methods that include electrochemical-based detection,14
16 nanopores,17
21 and CMOSbased devices.22 Methods for analysis of gene expression and for multi species DNA/RNA screening are also of high demand. For these applications, polymerase chain reaction (PCR)-based techniques have been highly instrumental.23,24 Despite the significant advances in DNA/RNA detection methods, there is still a need for simple and mobile technologies that exhibit comparable or improved sensitivity and can detect and identify both DNA and RNA simultaneously. For many applications, sensitive detection of a few molecules, either DNA or RNA, can provide such an advance in diagnostic technology as well as for studies in cell biology. A new method for DNA and RNA detection and identification is presented here. This method is sensitive, fairly simple, and can r 2011 American Chemical Society

detect both DNA and RNA simultaneously without pretreatment. It is based on the ability to pattern the inner surface of a capillary tube with oligonucleotide probe molecules in welldefined locations and subsequently flowing a solution containing the analyte DNA, RNA, or both through the tube. Upon detection of appropriate DNA and RNA from the sample, DNA polymerase and reverse transcriptase (RT) are employed to synthesize the complementary nucleic acids with deoxynucleoside triphosphate (dNTP) labeled with fluorophores. The formed hybrids are sensitively detected by their fluorescence signal.

’ MATERIALS AND METHODS Figure 1 describes schematically the detection process of DNA (a) and RNA (b). Tube Preparation. A concentration of 1 mM of aminopropyltrimethoxysilan (APTMS) solution is the first building block connecting the probe molecules to the surface. It was injected into 1.5 cm long, 0.3 mm i.d. glass tubes with a volume of 1 μL and incubated for 2 h at room temperature. Next, the tube was washed with ethanol and dried with nitrogen. A 1 mg mL
1 Received: August 4, 2011 Accepted: October 31, 2011 Published: October 31, 2011 9418

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Figure 1. (a) A scheme describing the detection of DNA and amplification of the signal by using DNA polymerase and dNTPs, which include fluorescent dCTP. (b) A scheme describing the detection of RNA and amplification of the signal by using reverse transcriptase and dNTPs, which include fluorescent dCTP.

solution of NHS-biotin, the second building block, was injected into the tube and reacted with the amino functional patterned surface for 1 h. The tubes were then washed with ethanol, dried with N2, and washed with water. Then, 10 μM streptavidin, with four binding sites for biotin, was immersed into 50 mM phosphate buffer (pH 7.5) and injected into the tube (reactivity units of streptavidin at this concentration were 14 units mL
1). The tube was incubated for 1 h at room temperature and then washed with 50 mM phosphate buffer (PB). In this stage, a control check, using fluorescently labeled streptavidin, was conducted to reassure that the above complex was evenly distributed on the surface. DNA probes were chemically attached to the inner surface of the tube using an 80-base oligonucleotide modified with biotin at the 50 end (all sequences used are numbered and detailed at the end of this section). The DNA probe was designed to include two regions: a 20-base segment that is complementary to the desired analyte DNA at the 30 end and a 60-base region enriched with guanine residues (a total of 15 bases) at the 50 end. The 30 region is used as the hybridization anchor for the analyte DNA, whereas the 50 region is used as a template for signal amplification by the DNA polymerase. To immobilize the DNA probes (1), 50 μM of the biotin-modified probe were immersed into 50 mM PB (pH 7.5) and injected into the tube. The tube was incubated for 1 h at room temperature and then washed with 50 mM PB. Figure 1S in the Supporting Information presents the scheme used for the tube’s internal surface patterning process.

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The probe molecules are patterned through an eight-stepautomated procedure of the magneto-lithography method,25,26 described in detail in the Supporting Information. Each patterned site is 0.8 mm wide. While in the present study the tube was patterned with up to two sites, in principle it can be patterned with several sites. All oligonucleotides were purchased from Eurofins Company and purified by HPLC. DNA polymerase (Klenow I Fragment) and Reverse Transcriptase (RT) (SuperScript II) were purchased from Bio Lab and Invitrogen, respectively. Deoxy adenosine triphosphate (dATP), deoxy thymidine triphosphate (dTTP), deoxyguanosine triphosphate (dGTP), and 5-propargylamino deoxy cytosine triphosphate-acridine orange dye (dCTP-ATTO-495) were purchased from Jena Bioscience. The DNA and RNA sequences used were (1) DNA oligonucleotide probe, biotin50 ATT GCC TGA ATG TAC GTC TGA AAG CCT GTT GAT GCC TGA ATG TAC GTC TGA AAG CCT GTT GAC GAT GGA AGG GAA AAC AG30 ; (2) DNA oligonucleotide analyte, 50 CTG TTT TCC CTT CCA TCG TC30 ; (3) RNA oligonucleotide analyte, 50 GCC CGA ACG AAG ACA GCC CGC UGU UUU CCC UUC CAU CGU C30 ; (4) total RNA was extracted from Neurospora crassa as described by Ziv et al.;27 (5) total RNA was extracted from tomato (Lycopersicon esculentum). DNA Detection. The oligonucleotide analyte DNA used is an artificial, 20 base long, single stranded DNA (ssDNA) (2) that was dissolved in 100 mM PB (pH 7.5). Hybridization between the DNA probe and the analyte occurred after injecting 1 μL of analyte solution into the tube containing the appropriate DNA probe. The tube was incubated for 1 h at 50 °C and then washed with PB solution. For signal amplification, we used a mixture of PB solution containing 10 units mL
1 DNA polymerase I, Klenow Fragment enzyme with 10 μM of dNTP, and 50 μM of dCTP labeled with ATTO 495. A volume of 2 μL of this mixture was injected into the tube and incubated for 90 min at 42 °C. Subsequently, the tube was washed with PB and dried with nitrogen. The emission signal was measured, at 527 nm, by using a fluorescence microscope (Olympus BX62 and BX50WI). RNA Detection. A 40-base artificial RNA oligonucleotide (3) or total RNA extracted from N. crassa (4) or total RNA extracted from tomato (5) was dissolved in 100 mM PB (pH 7.5) (Figure 1b) (all sequences are detailed and numbered at the end of the tube preparation paragraph above). A reaction solution containing 20 μL of the analyte RNA, 200 units of reverse transcriptase, 0.1 M dithiothreitol, 10 μM of dNTP, and 50 μM of dCTP labeled with ATTO 495 was prepared. A volume of 1 μL of this mixture was injected into the tube and incubated for 90 min at 42 °C. Then, the tube was washed with PB and dried with a stream of nitrogen. The emission signal was measured as described above for DNA detection. Detection of native mRNA was tested using a 20-base probe derived from the actin gene (NCU04173.4) sequence of the filamentous fungus N. crassa.28 The entire length of the N. crassa actin mRNA is 1448 bases, and the sequence used for detection corresponded to nucleotides 296 to 316 of the mRNA. This implies that 296 bases were potentially used as a template for RT polymerization. This sequence of 296 bases includes 48 guanine bases, which can bind to fluorescent cytosine. Signal Amplification Method. Once the analyte is bound to the probe, the double-stranded nucleotide chain serves as a priming site for the 50 to 30 extension, incorporating fluorescent 9419

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Figure 2. The fluorescence signal intensity of a DNA analyte as a function of the number of DNA molecules injected, spanning a concentration range of 12 orders of magnitude. The insert shows the fluorescence intensity for injection of a constant number of molecules. This was achieved by injecting increasingly larger volumes of solution of appropriately decreasing concentrations at a constant injection rate of 40 μL min
1. Also shown are images of the fluorescence signal for various DNA concentrations. The error bars represent 1 standard deviation.

nucleotides from the reaction solution. Following the appropriate washes described above, the resulting chromophorecontaining chain can be detected and quantitatively analyzed by standard fluorimetric methods. For detecting the fluorescence signal from the tube, the tube was photographed by a fluorescence microscope camera (Olympus BX62 and BX50WI). The image was obtained by collecting the emitted light at a 527 nm. The brightness of the image was calculated by integrating the signal over all pixels. This brightness is considered as the “signal” obtained.

’ RESULTS AND DISCUSSION Sensitivity of the PCT. The PCT sensitivity for DNA detection was determined using an 80-base-long probe that has two characteristics: (i) at the 30 end, a 20-base sequence complementary to the analyte and (ii) a 60-base-long segment used for signal amplification (see Figure 1a). The latter section included 15 guanine bases that complement the 5-propargylamino-dCTP in the developing solution. Hence, 15 chromophores attached to each probe
analyte complex (see the Materials and Methods). The detected fluorescence signal for injection of 1 μL of solution containing between 102 to 1014 analyte DNA molecules (equivalent to 10
16 to 10
4 M) is shown in Figure 2. The number of molecules detected was determined by appropriate dilution of a stock solution. The signal was linear for solutions with lower than 105 analyte molecules, and it is saturated at 108 molecules. The inset in Figure 2 further demonstrates the detection sensitivity to the number (rather than concentration) of molecules. Specifically, the same number of molecules was introduced into the tube, but they were dissolved in different volumes of solutions; thus, the concentrations differed. The total volumes were injected into the tubes at a rate of 40 μL min
1. After all solutions were injected into the tubes, they were incubated at 50 °C for 1 h. The signal intensity remained

nearly constant, even when the solution was diluted by 4 orders of magnitude. Figure 3 shows the analysis of at least three samples with the lowest number of analyte molecules (ranging from 1 to 600 molecules). On the basis of the calibration curve, as well as the statistics of the signal, we determined a detection limit of a few molecules. This experiment clearly indicates that the PCT can detect a very low amount of analyte molecules independent of their concentration in the solution. The high sensitivity of this method is a result of the small inner diameter of the tube. The diffusion time to the walls of the tube is about 10 s for a 100 μm inner diameter tube, which is substantially shorter than the residence time in the tube. This calculation assumes a diffusion coefficient for the DNA single strands of about 55 μm2 s
1. Thus, the ratio between the flow rate and the diffusion time ensures that all analyte molecules can interact with the probe-coated walls. RNA detection was studied by designing and synthesizing a 40-base-long RNA that was used as the analyte. This analyte had two sections: (1) a 30 end region of 20 bases that complement the probe (detector) sequence and (2) an additional section of a 30 end region of 20 bases which is used as a template for elongation (see Figure 1b). The second section included six guanine bases, designed to incorporate six cytosine fluorescent bases into the probe strand during RT polymerization. Hence, six fluorescing chromophore units were bound in every analyte-probe hybridization event. The fluorescence signal obtained, as a function of the RNA analyte concentration, is shown in Figure 4a. The signal spanned a range of 10 orders of magnitude in the RNA analyte concentration until reaching saturation in the presence of 1010 analyte molecules. The sensitivity for native mRNA detection was tested with an 80-base-long probe, where the 20 bases at the 30 end complement N. crassa actin mRNA. A solution of 10
11 M (10 7 molecules in 1 μL) total N. crassa mRNA, which is within the linear response range of the device (Figure 4a), 9420

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Figure 3. Fluorescence signal recorded from a PCT for 1-700 DNA molecules (by increasing the injected volumes). In the insert, the signal from the injection of 1
30 analyte molecules is shown. The concentration of the solution was determined by injecting 1 μL of the diluted stock solution.

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Figure 5. Comparison between PCT and RT-PCR. In blue, the RTPCR results, where CT is the number of threshold cycles needed for detection. The mean CT value was calculated from the triplicate reaction and is expressed by 2
CT. In black are the PCT results. The fluorescence signal is represented by arbitrary units. The lines are for presentation only.

Figure 4. (a) Concentration-dependent curve of a RNA analyte and (b) time-dependent fluorescence signal of 10
11 M N. crassa RNA analyte.

Figure 6. (a) Fluorescence signal of a sensor exposed to a 1 nM RNA solution from tomato and a mixture sample of 10 pM RNA from N. crassa with 1 nM RNA from tomato. (b) Concentration-dependent curve of sensing a N. crassa RNA analyte in a mixture containing human serum.

was injected; the fluorescent signal saturated after ∼20 min (Figure 4b). Further tests were conducted by comparing the new PCT detector to real-time PCR (RT-PCR). Figure 5 presents a

comparison for the same N. crassa mRNA sample using the two methods. The PCT sensitivity was equal to, or better, than that of RT-PCR, reaching a detection sensitivity of a few molecules. 9421

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Figure 7. Demonstration of specificity and selectivity of a PCT, using a PCT patterned for both DNA and RNA detection. A DNA analyte sample was injected into the tube, followed by a “developing” solution containing DNA polymerase with dNTPs. The DNA probe site fluoresces, whereas the RNA probe site remains dark. Following injection of RNA analyte and a developing solution containing RT with dNTPs, the RNA site also fluoresces. A reciprocal experiment is also presented. The fluorescence intensity (in arbitrary units) is presented under each image.

Specificity and Selectivity of the PCT Detector. Chemical processes on surfaces may suffer from reduced selectivity due to nonspecific interactions of the analyte with the surface. Interference occurs if the nucleotides in the solution interact nonselectively with the tube’s surface. To investigate the selectivity and specificity of the PCT sensor, N. crassa total mRNA was mixed with a 100-fold higher concentration of total mRNA from tomato (10 pM RNA from N. crassa mixed with 1 nM RNA extracted from tomato). The probe used was designed specifically for N. crassa actin mRNA. The specific-to-nonspecific signal ratio was about 10:1 (Figure 6a), and since the concentration of the nonspecific mRNA in the preparation is a hundred fold higher than that of the specific one, we conclude that the specificity of the detection was greater than 1000:1. The sensitivity and selectivity of the detection was further demonstrated when the N. crassa total mRNA was mixed with a human serum sample, which did not interfere with the detection of the specific mRNA (Figure 6b). The selectivity is also demonstrated in Figure S3 in the Supporting Information. It is possible to use PCT without special sample preparation and to characterize both DNA and RNA from the same sample. Selectivity is obtained by appropriate design and application of the “developing solution”; to first include an enzyme that elongates one type of oligonucleotide (i.e., DNA polymerase) and subsequently injecting another developing solution that contains the enzyme required for a different elongation process (i.e., RT). This is demonstrated in Figure 7, with a tube patterned with dual-probes: one for DNA and one for RNA. An analyte solution containing 1 pM of DNA was first injected through the tube, followed by a solution containing DNA polymerase and dNTPs. At this stage, only the DNA probe was elongated, whereas the RNA probe did not incorporate fluorescent chromophores. After injecting a solution of 1 pM RNA and addition of RT with dNTPs, the RNA probe was elongated and fluorescence was observed at the two sites. The reciprocal experiment

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was performed with another patterned tube and showed similar behavior Our results indicate that PCT is highly specific even in the presence of potentially interfering elements, as shown in the measurements performed on mRNA in the presence of human serum without the need for pretreating the analyte solution (Figure 6b). In PCR, in contrast, the presence of serum that contains proteins and other residues may interfere and reduce the detection sensitivity and selectivity.29,30 PCT also offers a simple calibration method. By calibrating the signal of the fluorescence intensity, it is possible to detect and quantitatively analyze DNA/RNA sequences within a large dynamic range, from nanomoles to a few molecules. Preparation of the Analyte Sample. The lack of pretreatment in the PCT method offers several potential advantages over other methods used for similar applications. In methods based on two-dimensional surfaces probes, such as microarrays, the sample preparation requires a multistep pretreatment that involves extracting and purifying the nucleic acid as well as amplifying and labeling the analyte before it is introduced to the detector. Furthermore, when investigating mRNA by microarrays, an additional step is required for translating the mRNA to cDNA. This is eliminated in PCT as there is no need for purifying or amplifying the sample, since the mRNA analyte is attached to the probe as is and the elongation of the analyte sequence along with the formation of a labeled cDNA (the signal) are done in situ with the use of RT. Detection Time. In methods based on probes immobilized on a two-dimensional surface, the detection time is inversely proportional to the concentration of the analyte, because the analyte molecules must diffuse on the surface until they interact with the probe “pixel”. In PCT, the analyte flows through the tube and the relevant diffusion time is related to the diffusion in the transverse direction. Hence, tubes with smaller diameters provide a higher ratio between the surface area of the probe and the volume of the analyte solution and facilitate faster diffusion, which leads to a faster detection time (ranging from 5 to 30 min). The short time and the high sensitivity of the system result from the fact that the entire sample is flowed through the tube. It is possible to combine several PCT tubes in a microfluidic setup that allows, for example, circulating the analyte solution several times through the tubes to ensure detection of even minute amounts of molecules. A number of probes can be interconnected in an arrayed structure so several detector tubes can analyze the same analyte solution. This configuration is analogous to a two-dimensional array, but the analyte solution flows in series from one “pixel” to another, significantly reducing the required detection time. It is expected that PCT will cost less than real-time PCR machines, due to its simplicity.

’ CONCLUSIONS We described a new method for ultrasensitive detection of both DNA and RNA by PCT. We determined that PCT has several important advantages; it has a very low detection limit of just a few molecules and it can detect both DNA and RNA simultaneously. The PCT is highly specific even in the presence of potentially interfering elements, such as human serum or nontarget nucleic acids. Furthermore, no pretreatment of the analyte solution is needed, and once the sample is inserted into the tube, the diffusion time of the analyte is fairly fast, due to the 9422

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Analytical Chemistry small diameter of the tube. This enables the high sensitivity and short detection times. The patterned tube can be produced by a simple and automatic procedure, and therefore tubes patterned with several different probes are simple and inexpensive to produce. The detection part of the system is also simple and compact, mechanically stable, and mobile. It can be made from a series of light emitting diodes and a strip of CCD elements. The current study shows that capillary tube patterning in a simple way opens up the possibility of quantitative detection and identifying DNA/RNA molecules in a new, sensitive, simpleto-operate, and inexpensive detection system. The simplicity of the detection may enable new applications of DNA and RNA analysis in medicine and environmental studies.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT A.B. and N.B contributed equally to this work. R.N. and Y.R. acknowledge the support of the Grand Center for Sensors and Security at the Weizmann Institute. Y.R. acknowledges support by the Helen and Martin Kimmel Award for Innovative Investigation. O.Y. acknowledges the support of the Israel Science Foundation. ’ REFERENCES

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