Anal. Chem. 1993, 65, 849-852
849
Fluorescence Detection and Size Measurement of Single DNA Molecules Alonso Castro, Frederic R. Fairfield, and E. Brooks Shera’ Biophysics and Theoretical Biology Groups, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
We have developed a technique for the detection and dze dlscrlmlnatlon of single DNA molecules In a hydrodynamlcally focused flowing solutlon. Double-stranded X DNA molecules at 3 X lo-” M were stained with the fluorescent dye TOTO-1 and were lndlvldually detected. The technique makes use of a frequency-doubledmoddocked NdYAG laser to repetitively excite the molecules as they traverse the tightly focused laser beam. The flowing sample solution was hydrodynamically focused down to a 20-pmdlameter stream by a rapidlyflowing water sheath. The sheath flow technique Is well suited for laser-lnduced fluorescence detectlon of small-volume, lowconcenlratlonsamples. The emitted fluorescencephotonbunt origlnatlng from a dngle DNA molecule was detected with a microchannel plate phoromultlplkr based slngleghoton cwrter, whlch used tlme-gated electronlcs for Raman and Raylelgh scattering rejection. I n addition, a mixture of A DNA and smaller singlacut fragmentshas beenslmuitaneoudydetected and ldentlfled by slze. The advantages over other techniques for the detection and slze determination of DNA fragments are discussed.
INTRODUCTION The detection and identification of minute amounts of nucleic acids is required in many fields, such as molecular biology, biotechnology, medical diagnostics, and forensic analysis. Radioactive labeling is the most widely used technique for the detection of trace amounts of DNA,1+2 primarily because of its sensitivity, which extends down to the picogram range. However, the difficulties associated with the lifetime, handling, and disposal of radioactive reagents have created an interest in alternative detection strategies. Recently, for example, Rye et al.3 have developed a method for the detection of DNA in agarose gels based on fluorescence emission. This method has a sensitivity of 4 pg per band, which approaches that of radiolabeling methods. Nevertheless, many applications require the detection of even smaller
* Author to whom correspondence should be addressed at Mail Stop D434. (i) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning, 2nd ed.; Cold Spring Harbor Laboratory Press: New York, 1989; pp 6.21, E.21. (2) Maxam, A. M.; Gilbert, W. Methods Enzymol. 1980,65,499-559. (3) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992,20,2803-2812.
samples of DNA. Currently, the polymerase chain reaction technique (PCR)4,5is widely used to amplify specific DNA sequences, making detection more feasible. Although PCR is a highly effectiveamplification mechanism, the use of many PCR cycles may introduce ambiguities arising from contamination and by mechanisms not yet fully understood.”7 It is for these reasons that it is important to develop more accurate, sensitive, and faster techniques for the detection of small amounts of DNA. In this paper, we describe the use of our recently developed technique of single molecule detection879 to efficiently detect single DNA molecules in a hydrodynamically focused flowing solution. The technique involves repetitive laser excitation of individual molecules,detection of the emitted fluorescence light with a microchannel plate photomultiplier based singlephoton counter, time-gated electronics, and signal processing. The signature of the passage of individual molecules is a burst of photons that occurs as the molecule traverses the laser beam. Since the fluorescence quantum yield of DNA is very small, we use a modified version of the staining technique used by Hirschfieldlo in his early observations of individual protein molecules. In the present experiments we stained the native DNA with the fluorescent dye TOTO-111 (a dimer of thiazole orange). TOTO-1,which intercalates between the DNA bases, has a binding affinity constant nearly lo00 times larger than that of the most widely used DNA intercalator, ethidium bromide.l2 Also, when bound to DNA, the TOTO-1-DNA complex fluorescence quantum yield increases by a factor of 1100 compared to that of free TOTO-1.3 This makes it an excellent candidate for ultrasensitive detection of DNA molecules. In the present experiments, we demonstrate the detection of full length duplex X phage DNA molecules (48502 base pairs). In addition, a mixture of X DNA and smaller single(4) Scharf, s. J.; Horn, G. T.; Erlich, H. A. Science 1986,233, 10761078. (5) Bej, A. K.; Mahbubani, M. H.; Atlas, R. M. Crit. Reu. Biochem. Biophys. 1991,26, 301-334. (6) Dunning, A. M.; Talmud, P.; Humphries, S. E. Nucleic Acids Res. 1988,16, 10393. (7) Kwoh, S.; Higuchi, R. Nature 1989,339, 237. (8) Shera, E. B.; Seitzinger, N. K.; Davies, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553-557. (9) Soper, S. A.; Davies, L. M.; Shera, E. B. J.Opt. SOC.Am. B. 1992, 9,1761-1769. (10) Hirshfield, T. Appl. Opt. 1976, 15, 2965-2966. (11) Molecular Probes, Inc., Eugene, OR. (12) Huang, Z., Molecular Probes, Inc., personal communication.
This article not subject to U S . Copyright. Published 1993 by the American Chemical Society
850
ANALYTICAL CHEMISTRY, VOL. 65, NO. 7. APRIL 1. 1993 SPATIAL
ATTENUATOR CYLINDRICAL LENS CYLINDRICALLENS
MULTICHANNEL SCALER
I
ADC
I
-,n, WORKSTATION
68030CPUCAMACCRATE VMEI CRATE CARD
sheath lluid
A schematic drawing of the apparatus we have used for the detection of single molecules is shown in Figure 1. A SpectraPhysics 3800 frequenry-doubled mode-locked NdYAC laser producing 7 0 . ~ 3pulses at 532.nm wavelength and 82-MHz repetitionratewasusedas theexcitationsource. The laseroutput was directed through a variable neutral density filter whirh attenuated thelaserintensity to5(t;OmW. Thelaser beamwus focused into the sample cell by a pair of crossed cylindriral lenses to yield a 5M) X 10-pm spot (horizontal and vertical I e- values, respectively). This arrangement provided uniform excitation along the entire cross section of the hydrodynamically focused samplestream (see helowi. The sheath flow cell wasconstructed from a glass square-bore capillary with inside dimensions of 800 X 8W pm (Figure 21. The sample was gravity fed through a 100-rrm-diameter stainless steel hypodermic needle into the square cell. A sheath of water wan pumped hy a Harvard Apparatus Model 22 syringe pump through a 0.1-um Millipore filter and into the square cell at about 0.8 mL h. This rapidly flowing water shenth hydrodynamically focused the sample streamtoafinaldiameterof20~m. 'hecombined useofalarge horizontal beam dimension and a small stream diameter should permit homogeneous illumination of the sample, such that all molerules experience identical laser excitation intenities. This modification of the rightly focused Gaussian laser beam profile used in previous experiments" was required if meaningful fluurescence intensity measurements are M be made. The detection volume created by the intersection of the laser beam and the fcrused sample stream was 3 pL. The sheath flow technique'? is well suited for laser-induced fluorescence of low. concentration samples because of the small prohing volume produred. which provides low barkground fluorescence and uniform excitation of the sample. Fluoresrent light wascollected by a 40x 0.65 N.A. microscope ohjective, spatially filtered by a (13) Zanin,
F.: Dovirhi. N. J. Anal. C h m . 1985.57.269CF2692.
h
1 r J IbW
cell
./ wall
Flgure 2. Cross Section of the sheath flow cell (not to Scale). The sheath IIquM flows through a glass squarebore cell (0.8mm 1.d.) and hydrodynamically focuses the Sample stream from 100-pm4.d. Iniectbn tube down to 20 am. The laser beam travels perpendlcular to the drawing plane. 1.2- x 0.4-mm rectangular slit, spectrally filtered by a 555/30 nm eight-cavity Omega Optics interference filter, and detected by a Hamamatsu R1562U microchannel-plate photomultipliertube cooled t o 0 OC. The MCP-PMT signal wan amplified by a HewlettPackard 8447F amplifier, shaped by a Tennelec TC454 discriminator and sent t o the gate and stop inputs of a Tennelec TC863 time t o amplitude converter (TAC). A fraction ofthe laser beam was split off by a beam splitter and sent to a fast photodiode to provide the start pulses for the TAC. A time-gate window was set such that both prompt Raman and Rayleigh scattering were rejected, and only delayed fluorescence photons were detected.* Events occurring during the time window were counted by a CAMAC Joerger S3 multichannel scaler. The CAMAC bus was interfaced via a CES Model CBD-8210 V M E interface to a Sun workstation. The data stream from the scaler was stored on disk
ANALYTICAL CHEMISTRY, VOL. 65, NO. 7, APRIL 1, 1093
-
300
4
DNA + TOTO-1 in TE buffer
I
851
I
3? 250
P
$
*0°
a;a
150
E
;100 U L
;
I
20b0
4000
6 .-
50
5
100
6000
Time (picoseconds)
Figure 9. Tlmedeiay histograms for a 2 X 10-l2 M soiutlon of DNATOTO-1 complex in TE buffer (upper curve) and for a similar solution wlthout DNA, Le. TOTO-1 In TE buffer (lower curve). The position of the timegate wlndow used to reject Raman and Raylelgh scatterlng Is shown.
for off-line analysis and was also analyzed in real-time using a VME-bus color monitor driven by the Sun computer. This displayprovides a chart-recorder-stylegraph of the photon bursts that occur as individual DNA molecules pass through the laser beam, thus facilitating rapid experimental optimization. Determination of the sample stream flow velocity was accomplished in a separate measurement by observation of 1-pm fluorescent microspheres as they flowed through the detection volume. A microscope objective imaged the detection volume into a CCD video camerawhose output was recorded and analyzed frame-by-frame. The linear flow velocity was determined in this way to be 700 fim/s,which corresponds to a transit time through the laser beam of about 20 ms. A similar procedurewas employed to determine the sample stream diameter, which was considered to be the same for microspheres and DNA, since the effects of diffusion are negligible in both cases under our experimental conditions. X DNA (full-length and Xba I digest) and TOTO-1 were purchased from New England Biolabs (Massachusetts) and Molecular Probes (Eugene,OR), respectively. Sample solutions were prepared by diluting the X DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to the desired concentration. TOTO-1 was added to yield a final base pair to dye ratio of 5:l. Water was deionized and doubly distilled prior to use, and all solutions were prepared immediately before each experiment.
RESULTS (a) Time-Gate Optimization and Estimation of the X DNA-TOTO-1 Complex Fluorescence Lifetime. In order to determine the position of the time-gate window for best rejection of Raman and Rayleigh scattering, a time-delay histogram of the TAC output was accumulated for a 2 X 10-12 M X DNA solution in T E buffer to which TOTO-1 was added a t a base pair to dye ratio of 5:l. The results of this measurement are depicted in Figure 3, where the position of the selected time-gate window is shown. The fluorescence lifetime was determined to be approximately 1.7 ns. Also shown in Figure 3 is the time-delay histogram for a TOTO-1 solution in T E buffer at the same concentration, but without X DNA (the prompt peak due to Raman and Rayleigh scattering is clearly evident). A comparison of the two curves indicates the large enhancement in TOTO-1fluorescenceupon intercalation into DNA. (b)Detection of Single Full-Length X DNA Molecules. To demonstrate the detection of individual DNA molecules, we used a flowing sample consisting of 3 X 10-15 M X DNA molecules (48 kbp) stained with TOTO-1at a base pair to dye ratio of 5:1 in T E buffer. At this concentration, the probability of a DNA molecule occupying the detection volume is 0.004. The probability of two molecules occupying the detection volume at the same time is then 1.6 X 10-5. Thus, the detection of two or more DNA molecules a t the same time is unlikely. The results of the experiment are shown in Figure 4. Each
50 0
2
4
6
8
1
0
1
2
Time (sec)
Figure 4. Welghted sum plots for a 3 X 10-15 M solution of DNATOTO-1 complex in TE buffer (top) and for a simliar solution without DNA, la., TOTO-1 in TE buffer (bottom).
observed peak is the integrated photon burst emitted by an individual DNA molecule as it traverses the laser beam. An average of 400 photons per X DNA molecule were obtained. The total number of detected molecules, N,for the 819.2-8 data stream was 346. This quantity is in agreement with the estimated value of 326, as calculated by the equation
N = cuvT where c is the concentration, u is the sample stream crosssectional area, u is the stream flow velocity, and T is the duration of the experiment. The comparison of the observed and computed number of DNA molecules indicates that the detection efficiency is 100% within our estimated experimental error. For better visualization of the photon bursts, the data stream was subjected to a weighted sum algorithm given by k-1
where k covers a time interval of the order of the molecular passage time, d(t + T ) is the data point at time t T , and W ( T ) are weights that resemble the photon burst shape (a doublesided symmetrical ramp was used in the present case). The experiments were also performed under identical conditions with asimilar sample solution except that DNA was not added, Le., TOTO-1 in T E buffer at the same concentration. The resulting data stream is shown a t the bottom of Figure 4. No photon bursts were detected in this case. (c) Simultaneous Detection of X DNA and X Xba Digest. In a second series of experiments, a mixture of X DNA (48 kbp) and X DNA Xba I digest was used. The Xba I digest of X DNA yields two fragments of about 24 kbp each. The concentration of the mixture components was adjusted to obtain a 1:lratio of large (48kbp) to small (24 kb) fragments at 3 X M each. Since the amount of intercalated dye is proportional to the DNA size? one would expect to observe fluorescent molecules of two different brightnesses corresponding to the two DNA fragment sizes. This is indeed the case, as shown by the histogram plot of the number of peaks vs peak amplitude in Figure 5. The distribution centered at about 150 intensity units corresponds to 24 kb DNA pieces, and the one centered at 300 corresponds to 48 kb DNA pieces, yielding the expected 2:l intensity ratio. Thus, we have been able to quantify the detection of DNA molecules of different sizes with a resolution of 12-15 % ,as determined by the widths of the histograms in Figure 5. Once again, the number of detected molecules was in agreement with the calculated value: 651 vs 640, respectively. We believe that the size resolution in the present measurements is limited primarily by two factors: (1)statistical
+
852
ANALYTICAL CHEMISTRY, VOL. 65, NO. 7, APRIL 1, 1993
0
100
200
300
400
500
Peak height (fluorescence intensity)
Flguro 5. Histogram of the number of molecules vs fluorescence intensity for a mixture of h DNA (48 kbp) and h DNA Xba digest (24 M each. kbp) at 3 X
fluctuations in the number of photons detected from individual molecules (shot noise) and (2) lack of uniformity of laser illumination across the sample stream caused by imperfections in the glass capillary and by diffraction effects.
DISCUSSION The present results suggest techniques that may be useful in developing biotechnological,clinical, and forensic methods that do not require extensive DNA amplification using PCR or other methods. As mentioned, the sensitivity limit for fluorescence detection of DNA in agarose gels is of the order of 4 x 10-12 g/band.3 A similar sensitivity can be achieved with radioactive labeling methods. In the present case, the detection and size identification of a single 24 kbp DNA (14) Steen, H. B. In Flow cytometry and sorting, 2nd ed.; Melamed, R. M., Lindmo, T., Mendelsohn, M., Eds.; Wiley-Liss: New York, 1990; Chapter 29.
fragment translates into a sensitivity limit of 3 x 10-17 g. However, the size resolution of gel electrophoresis can be only a few percent or less, depending on the fragment sizes being separated, and may be as small as 0.2% for sequencing gels, whereas our technique has a current size resolution of 12-15 % . Nevertheless,in many cases,only a few DNA lengths may be present in a sample, and high resolution is not required. We are currently working to improve the resolution of our technique to differentiate among DNA fragments which differ in size by a few percent. For comparison, another technique which uses fluorescence emission for the detection of DNA is flow cytometry. Determination of the DNA content of a single Escherichia coli bacteria has been reported using this technique.14 However, the DNA content of the E. coZi genome is about 4 X lo6base pairs, more than 100 times greater than that of the single fragments detected in the present experiments. The sensitive detection of other individual biological molecules has important applications. Our current single fluorophore detection limit allows us to study solutions of concentrations in the sub-femtomolar range. Potential applications include the detection and measurement of low levels of pollutants or toxins, and the study of hormones directly at their biologicallyactive levels of 10-12 M and below. Currently, these concentrations are only detectable through indirect biological means.
ACKNOWLEDGMENT The authors would like to thank Lloyd Davis, Carol Harger, and Steven Soper for helpful discussions and Mark Peters for technical assistance.
RECEIVEDfor review November 10, 1992. Accepted January 2, 1993.
