Ultrasensitive Fluorescence Detection of Single Protein Molecules

electrically modulated fluorescence detection method for a protein assay with sensitivity and specificity down to the single molecule level. The targe...
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NANO LETTERS

Ultrasensitive Fluorescence Detection of Single Protein Molecules Manipulated Electrically on Au Nanowire

2008 Vol. 8, No. 9 2829-2833

Suxian Huang† and Yong Chen* Department of Mechanical and Aerospace Engineering, California NanoSystems Institute, UniVersity of California, Los Angeles, California 90095 Received May 18, 2008; Revised Manuscript Received June 25, 2008

ABSTRACT Proteins assembled on an Au nanowire are manipulated by an electrical potential applied on the nanowire, which leads to the modulation of molecular fluorescence. The molecular modality can be unequivocally correlated with the modulated fluorescence, which enables the specific fluorescence from a single target protein to be unambiguously distinguished from background noise and nonspecific fluorescence. As demonstrated through a thrombin assay, this simple method can significantly improve the sensitivity and specificity of the protein detection down to the single molecule level.

Recent advances in ultrasensitive protein biosensors have brought significant impacts to proteomics, biomedical diagnostics, and drug discovery.1-4 Advanced nanoscale biosensors based on nanoparticles, nanowires, and other nanomaterials have been developed to detect various proteins with improved sensitivity, specificity, and reliability.5-8 Ultrasensitive fluorescence nanosensors can detect the fluorescence signal from a fluorescence tag bound specifically with a single target molecule,9 but the plain fluorescence intensity measurement can hardly discriminate against a nonspecifically bound tag. Advanced fluorescence protein detection techniques such as fluorescence resonance energy transfer (FRET)10,11 and fluorescence correlation spectroscopy (FCS)12,13 have been used to interrogate fluorescence signals correlated with molecular modalities. However, it is still challenging to efficiently exclude false signals from nonspecific fluorescence, which often limits the sensitivity and specificity of protein assays. In this letter, we report an electrically modulated fluorescence detection method for a protein assay with sensitivity and specificity down to the single molecule level. The target proteins are assembled on an Au nanowire, and the specific fluorescence signal from the molecules is modulated while the molecules are manipulated by an alternating electrical potential applied on the nanowire. The simple method enables the regularly modulated fluorescence signal from the target proteins to be unambiguously distinguished from randomly fluctuated nonspecific fluorescence and noise. * Author to whom correspondence should be addressed. E-mail: yongchen@ seas.ucla.edu. Tel: (310) 206-2453. † E-mail: [email protected]. 10.1021/nl801429p CCC: $40.75 Published on Web 07/31/2008

 2008 American Chemical Society

In our electrically modulated fluorescence protein assay, an extensively studied protein, human R-thrombin, was chosen as the target.14,15 The scheme of our probe-targetreporter sandwich assay is shown in Figure 1a. A thrombinbinding DNA aptamer probe is assembled on a gold nanowire. The target, biotinylated thrombin, is captured by the aptamer probe, and then labeled by a fluorescent streptavidin reporter. It has been reported previously that short DNA oligos grafted on Au surfaces can be electrically manipulated by an electrical potential applied on the Au surfaces, resulting in the modulation of the DNA fluorescence.16,17 In our experiment, when an alternating electrical potential is applied on the nanowire, depending on the potential polarity, the negatively charged probe-targetreporter complex can be attracted or repelled by the nanowire, and the specific fluorescence from the complex is modulated accordingly due to the surface energy transfer (SET)18-21 between the fluorescent reporter and the Au nanowire. The Au nanowire was fabricated by depositing a 30 nm thick Au film on a 100 nm thick SiO2 layer on a Si (100) wafer, and a 60 nm thick SiO2 layer was sequentially deposited on the surface of the Au film by e-beam evaporation. After the wafer was cleaved along the Si (011) crystal planes, the cross-section of the Si wafer exposed a 30 nm wide gold nanowire sandwiched between the two SiO2 layers. The Au nanowires can be fabricated conveniently by this method with uniform nanoscale widths and extensive lengths, and they can also be electrically connected through the Au film reliably for electrical modulation. Compared with the Au thin film, the nanowire has a much smaller fluorescent

Figure 1. (a) A scheme showing an electrically modulated fluorescence protein assay including a thrombin-binding aptamer probe grafted on an Au nanowire, the target, a biotinylated thrombin, and the reporter, a fluorophore-labeled streptavidin. When a negative potential is applied on the nanowire, the negatively charged probe-target-reporter complex is repelled from the surface (left); when a positive potential is applied on the nanowire, the complex is attracted toward the nanowire (right). The fluorescence intensity from the probe-target-reporter complex is modulated as a function of the distance between the fluorophore and Au nanowire due to the surface energy transfer (SET). On the contrary, a reporter molecule nonspecifically bound to a surface site other than the target molecule is immobilized; neither its position nor fluorescence can be modulated by the electrical field. (b) Electrical potential applied on the nanowire (top) and modulated fluorescence measured from a sample at a 100 nM thrombin concentration (bottom) are shown synchronously versus time.

area, which can significantly reduce the scattering/reflection background noise, and enable the single molecule detection. The experimentally measured average fluorescence intensity per pixel from the nanowire is approximately 100 times stronger than the one measured from the Au thin film surface, probably due to the fluorescence signal enhanced by surface plasma generated on the Au nanowire.22,23 The chips were cleaned by piranha bath (H2O2:H2SO4 ) 1:3, 10 min), then soaked in the 1 µM solution of the thiolated thrombin-binding aptamer (Stanford PAN Biotechnology Facility, 5′ HS-(CH2)6-TTTCACTGTGGTTGGTGTGGTTGG 3′) in 1 M potassium phosphate buffer (pH 7.0) for 1 h to immobilize the probes onto the Au nanowires. Subsequently a thorough wash was performed with the potassium phosphate buffer to remove the unbound aptamers on the chip surfaces. The chips were then exposed to the target, biotinylated thrombin (EMD Chemicals) for 5 h at room temperature in thrombin-binding buffer (20 mM TrisHCl, pH 7.4 with 140 mM NaCl, 20 mM MgCl and 20 mM KCl) with thrombin concentrations ranged from 100 fM to 1 µM. After another extensive wash with the thrombinbinding buffer, the targets were reacted with the fluorophorelabeled streptavidin reporters (Invitrogen, Streptavidin@Alexa Fluor 546) for 30 min, followed by a final wash. To electrically manipulate the probe-target-reporter complex on the Au nanowire, the chip was immersed in an electrolyte (1 mM Tris-HCl, pH 7.3), and a square-wave (0.6 V alternating potential (Figure 1b) was applied on the Au nanowire at a frequency of 0.5 Hz with respect to an Ag/AgCl reference electrode and a Pt counter electrode through a standard three-electrode potentiostat (Bio-Logic SAS, Claix, France). The fluorescence was measured by a Nikon Eclipse E400 fluorescence microscope with a Y-2E/C filter (EX 540-580 nm, DM 595 nm, BA 600-660 nm). The fluorescence images were recorded by a fast-speed CCD camera (Photomatrix CoolSnap HQ2) as a function of time at a capture speed of 10 frames/s. Each pixel in a recorded 2830

fluorescence image corresponds to a ∼2.58 µm × 2.58 µm area on a chip. The typical modulated fluorescence signal from an image pixel on the nanowire is shown as a function of time in Figure 1b and Supplementary Movies S1 and S2 (Supporting Information). It was observed that when the positive potential was applied on the Au nanowire, the negatively charged molecule was attracted toward the nanowire, resulting in the increase of the SET from the fluorophores to the Au nanowire and the decrease of the fluorescence intensity accordingly. When the negative potential was applied on the nanowire, the molecule was repelled from the nanowire, and the fluorescence intensity increased (Figure 1a). Consequently the fluorescence was modulated synchronously to the alternating electrical potential. The fluorescence signals modulated at a certain frequency can be efficiently extracted by the so-called “lock-in” integration used extensively in electronics and optics.24,25 The detected fluorescence I(t) includes (i) the specific fluorescence from the reporter molecules specifically bound to the target proteins, IS(t); (ii) the nonspecifically fluorescence from the reporter molecules not bound with the targets, IF(t); (iii) the background noise, IN(t); i.e. I(t) ) IS(t) + IF(t) + IN(t). In our experiment, the specific fluorescence signal IS(t) is modulated synchronously by the alternating electrical potential oscillating at a frequency ω0. In the lock-in integration, I(t) is multiplied by a unit square wave function r(t,ω0) oscillating synchronously with the electrical potential and the modulated specific fluorescence IS(t) at the frequency ω0, and integrated over detection time T, i.e. Iˆ ) ∫T0 I(t)·r(t,ω0) dt/T. Neither the nonspecific fluorescence or the background noise is modulated by the alternating electrical potential, and they do not oscillate synchronously with the square wave function r(t,ω0), which leads to ∫0TIF(t)·r(t,ω0) dt ≈ ∫T0 IN(t)·r(t,ω0) dt ≈ 0. The lock-in integration output ˆI selectively preserves the specific fluorescence and filters out Nano Lett., Vol. 8, No. 9, 2008

Figure 2. (a) Fluorescence intensity jI (top) and lock-in integration output Iˆ (bottom) versus pixel position along an Au nanowire from a 100 fM thrombin sample. The type A pixels have a Iˆ value larger than the threshold value, and the type B pixels have a large jI value but a Iˆ value smaller than the threshold. (b) Pixel distributions of the fluorescence intensity jI and (c) pixel distributions of the lock-in integration output Iˆ along Au nanowires from a 10 pM thrombin sample and a negative control sample without thrombin. The Iˆ distribution from the control sample in (c) is fit with a bell-shape Gaussian distribution. The threshold value in (a) and (c) is set at four standard deviations (4σ) from the mean Iˆ value of the Gaussian distribution to distinguish between type A and type B pixels.

the nonspecific fluorescence and noise (Supporting Information). The modulated fluorescence signal from single molecules was detected from the samples with low thrombin concentrations (100 fM to 1 pM) in the buffer solution. The average fluorescence intensity, jI ) ∫T0 I(t) dt/T, measured from a 100 fM thrombin sample is shown as a function of the image pixel position along the nanowire in Figure 2a. The fluorescence signal I(t) from each pixel is processed by the lockin integration ˆI ) ∫T0I(t)·r(t,ω0) dt/T, and the integration output Iˆ is also shown accordingly in Figure 2a. Most of the pixels in the 100 fM thrombin sample have a low average fluorescence intensity jI close to the background noise level, and therefore contain no fluorescence. Among the pixels with fluorescence, two distinguishable types of pixels can be observed in Figure 2a: at the type A pixels, the lock-in integration output ˆI is high because the specific fluorescence from a probe-target-reporter complex is modulated synchronously by the electrical potential; at the type B pixels, jI is high but Iˆ is low, which represents nonspecific fluorescence that is not modulated synchronously by the electrical potential, probably from a fluorescent tag that is not bound with the thrombin. To quantitatively distinguish between the specific and nonspecific fluorescence, negative control experiments were performed with the same assay procedure but without the thrombin target. The fluorescence intensities from the pixels along the nanowire of the control sample and a 10 pM thrombin sample are measured, and the distributions of the average fluorescence intensities, jI ) ∫T0 I(t) dt/T, from the pixels of these two samples are shown in comparison in Figure 2b. The distributions of the two sets of the jI values overlap with each other, which indicates that from the fluorescence intensity measurement the specific fluorescence detected in the 10 pM thrombin sample cannot be distinguished from the nonspecific fluorescence and background Nano Lett., Vol. 8, No. 9, 2008

noise. We have also performed our thrombin assay with different thrombin concentrations ranging from 0 M (negative control) to 1 µM in the buffer solutions. The average fluorescence intensity per pixel is shown as a function of the thrombin concentrations in Supporting Figure S2 (Supporting Information), which indicates that the limit of detection (LoD) of the assay based on the fluorescence intensity measurement is ∼100 pM. After the fluorescence intensities from the 10 pM thrombin and control samples shown in Figure 2b are processed by the lock-in integration ˆI ) ∫T0I(t)·r(t,ω0) dt/T, the distributions of the lock-in integration outputs Iˆ from the pixels of the two samples are shown in comparison in Figure 2c. The Iˆ distribution from the control sample can be fit well by a symmetrical bell-shaped Gaussian distribution. A threshold value IˆT ) 2.0 is set at four standard deviations (4σ) of the distribution from the mean Iˆ value. The pixels with Iˆ < IˆT represent the nonspecific fluorescence or noise in the control sample. The Iˆ distribution from the 10 pM thrombin sample shows an asymmetrical “tail” from the pixels with Iˆ > IˆT, which indicates that these pixels have modulated specific fluorescence from the probe-target-reporter complexes and distinguishable larger Iˆ values than the nonspecific fluorescence and noise from the negative control sample. When a fluorescent reporter is immobilized on the surface or bound to other molecules rather than a thrombin, it cannot be effectively modulated by the electrical potential applied on the nanowire; therefore the nonspecific fluorescence has unambiguously distinguishable Iˆ value lower than IˆT. From the 100 fM thrombin sample, the pixels with specific fluorescence (“type A” pixels with Iˆ > IˆT in Figure 2a) can be explicitly distinguished from the ones with nonspecific fluorescence or noise (“type B” pixels with Iˆ < IˆT in Figure 2a). In the samples with a thrombin concentration less than 10 pM, the pixels with ˆI > IˆT only occupied a small fraction ( IˆT along the nanowire, and the numbers of the detected thrombins, NT, can be counted one by one. From all the detected single thrombin molecules, the average ˆI value per single thrombin molecule is derived as IˆS ) 529. In the negative control samples, no pixels have Iˆ > IˆT (since IˆT is set at 4σ value), therefore NT ) 0. When the thrombin concentrations were increased to 10 pM or higher values, the pixels with Iˆ > IˆT were increased and became partially or completely continuous along the nanowires, and it was likely for each pixel to contain more than one single thrombin molecule. In these cases, the average Iˆ value per pixel from each sample, IˆP, was measured and derived from the pixels with Iˆ > IˆT, and the average number of thrombin molecules per pixel was counted by dividing IˆP by IˆS, the average Iˆ value per single thrombin molecule. The average numbers of the thrombin molecules (NT) per pixel are derived from the Iˆ values as described above and shown as a function of the thrombin concentrations ranged from 0 M (negative control) to 1 µM in the buffer solution in Figure 3, which indicates that the thrombin assay has a LoD of 100 fM and a large linear dynamic range from 100 fM to 100 nM. The reproducibility of the assay was tested by measuring the data from two independent series of chips. As shown in Figure 3, the deviations between the two series of data are less than 10%. Compared with the LoD (100 pM) based on the fluorescence intensity measurement, the LoD based on the Iˆ measurement has been improved approximately by three orders. To the best of our knowledge, the LoDs of the previously reported aptamer-thrombin assays in buffer solutions are 1 nM by photoluminescence sensor,26 4 nM by ELISA,27 0.83 nM by colorimetric assay,28 5 nM by surface plasmon resonance (SPR) sensor,29,30 10 nm by electrochemical sensor,31 3 pM with DNA amplification and electrochemical sensor,32 2 pM with polymerase chain reaction (PCR) amplification,33 and 2 nM with rolling circle amplification (RCA).33 The improvement of our electrically modulated fluorescence assay is mainly due to 2832

the capability to eliminate the nonspecific fluorescence and background noise, which enable the single thrombin molecule to be unambiguously identified. From the titration curve (Figure 3), the binding affinity between the thrombin and aptamer was derived as ∼30 nM, which is within the previously reported ranges (25-200 nM).15 The number of the captured thrombin molecules can be calculated based on the binding affinity. The density of the DNA aptamer capture probes assembled on the Au nanowire is estimated to be 1012 sites/cm2. When a 30 nm wide Au nanowire is immersed in a 100 fM thrombin solution, it can be calculated that under the equilibrium condition only a small fraction (∼0.33 × 10-5) of the aptamers are actually bound with thrombins, which corresponds to ∼2.8 × 10-3 thrombin per imaging pixel under our fluorescence imaging condition. Based on our electrically modulated fluorescence assay, ∼1.6 × 10-3 thrombin per imaging pixel was detected, which is consistent with the theoretically estimated value. From the above analysis, we conclude that the LoD of the thrombin assay is mainly limited by the binding affinity between the thrombin and aptamer, and the LoD can be further improved by a target enrichment process. We also performed the thrombin assay in fetal bovine serum with the aforementioned procedures except (1) the serum was diluted in the thrombin-binding buffer (1:5 vol/ vol) to reduce the viscosity; and (2) 0.1% bovine serum albumin (BSA, Sigma) and 50 g/mL polyinosinic-polycytidylic acid potassium salt (poly dI/dC, Sigma) was added to the thrombin binding buffer to reduce the nonspecific binding of serum to aptamer. After the fluorescence intensities are processed by the lock-in integration, the distributions of the lock-in integration outputs Iˆ from the pixels of a control sample without thrombins and a 1 nM thrombin sample in the serum are shown in comparison in Supporting Figure S3 (Supporting Information), which indicates that the specific fluorescence can still be efficiently distinguished from the nonspecific fluorescence, and single thrombin molecules were unambiguously detected in the serum matrix. Based on the Iˆ values, the average numbers of the thrombin molecules (NT) per pixel are measured as a function of the thrombin concentrations ranged from 0 M (negative control) to 1 µM in the serum (Supporting Figure S4, Supporting Information). The LoD of the assay for thrombins in the serum is ∼1 nM, which is probably due to the blockage of the thrombin-aptamer binding by unknown matrix components in the serum.32 In summary, we have developed an electrically modulated fluorescence protein assay that can detect specific fluorescence from a single molecule assembled on an Au nanowire by manipulating the molecule with an electrical potential applied on the nanowire. As demonstrated through a sandwich protein assay, a target protein, human R-thrombin, was captured by a DNA aptamer probe grafted on an Au nanowire, and then labeled with a fluorescence reporter. By applying an alternating electrical potential on the Au nanowire, the probe-target-reporter complex was attracted toward or repelled from the Au nanowire, which modulated its fluorescence accordingly due to the SET between the Nano Lett., Vol. 8, No. 9, 2008

fluorescence reporter and nanowire. It was demonstrated that the molecular modality can be unequivocally correlated with the modulated fluorescence, which enables the specific fluorescence from a single thrombin molecule to be unambiguously distinguished from background noise and nonspecific fluorescence. Based on the electrically modulated fluorescence detection, the sensitivity and specificity of the thrombin assay can reach the single molecule level. The LoD of the assay in buffer solution is 100 fM, and the linear dynamic range of the assay can extend from 100 fM to 100 nM. The simple electrically modulated fluorescence detection method can be generally applied to various bioassays. The essential requirement of the method is to selectively modulate the specific fluorescence from the target molecules by an external reference field, which can be achieved by electrical, optical, magnetic, mechanical, or biochemical interactions etc. Acknowledgment. This work was supported by the U.S. National Institutes of Health (Grant AI065359) through Pacific Southwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, U.S. National Science Foundation under the Focus Center Research Program (FCRP)sCenter on Functional Engineered Nano Architectonics (FENA), and Center for Scalable and Integrated NanoManufacturing. We thank J. B.-H. Tok (Lawrence Livermore National Laboratory), X. Zhang and Y. Xiong (University of California, Berkeley) for valuable scientific discussions, and Z. Zhu, N. Jin, Y. Lei and C. Stuart (University of California, Los Angeles) for helpful technical inputs and review of the manuscript. Supporting Information Available: Electrically modulated fluorescence movies, lock-in electrically modulated fluorescence detection, titration curves based on fluorescence intensity measurement, pixel distributions of the lock-in integration output from samples in serum, and titration curves from the thrombin assays in serum. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293 (5537), 2101–2105. (2) MacBeath, G.; Schreiber, S. L. Science 2000, 289 (5485), 1760–1763. (3) Lee, S. J.; Youn, B. S.; Park, J. W.; Niazi, J. H.; Kim, Y. S.; Gu, M. B. Anal. Chem. 2008, 80 (8), 2867–2873. (4) Giuliano, K. A.; Taylor, D. L. Trends Biotechnol. 1998, 16 (3), 135– 140.

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