Gold nanoparticles as novel label for DNA diagnostics

Technology Report

Gold nanoparticles as novel label for DNA diagnostics Andrea Csáki, Robert Möller and Wolfgang Fritzsche†

CONTENTS

The growing interest in DNA diagnostics, especially in combination with the need for highly-paralleled and miniaturized hybridization assays, is today addressed by fluorescence DNA chips. Fluorescence detection is approved and highly developed, however, it has also problematic aspects, e.g., the low stability of the dyes, the influence of the physicochemical environment onto the signal intensity and the expensive set-up for detection. A novel detection scheme based on metal nanoparticles was proposed to overcome these problems and is discussed in this review.

DNA-nanoparticle complexes

Expert Rev. Mol. Diagn. 2(2), (2002)

Nanoparticle-labeled DNA on solid supports

DNA chips represent an emerging technology for parallel detection of DNA molecules, with applications ranging from environmental monitoring to diagnostics. The development of this technique is driven by the need for higher parallelization in combination with a decrease of sample volume, resulting in whole arrays of binding partners on a solid substrate. Especially for the highly integrated DNA chips, fluorescence labeling and detection became the method of choice, mainly due to the high sensitivity and the ease of use. However, the fluorescence intensity of the dyes depends on the physicochemical environment of the molecules, which hampers the needed quantification. Another problem is bleaching, the irreversible reaction of a dye leading to a loss of signal. Additionally, the detection of fluorescence signals needs a sophisticated optical set-up for filtering the exciting and emitting light. Reviewed here is a new labeling method based on gold nanoparticles. This method combines advantages of optical detection with novel and superior properties compared to the fluorescent dyes.

Assays based on glass slides Microstructured DNA-chips Single-particle detection Novel optical readout devices Summary & conclusion Expert opinion Five-year view Key issues References Affiliations

†Author for correspondence

Molecular Nanotechnology Group, Institute for Physical High Technology PO Box 100239, 07702 Jena, Germany Tel.: +49 364 120 6304 Fax: +49 364 120 6344 [email protected] KEYWORDS: DNA chip, gold nanoparticles, optical detection

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Gold nanoparticles are associated colloids, which can be synthesized with dimensions ranging from 0.8–250 nm [1]. They have been used extensively as a biomolecular label in light and

electron microscopy [2]; further applications for single electron tunneling devices or surface enhanced spectroscopy are under investigation. Extending the microscopical application of DNA-gold conjugates, structures of two or more colloids based on self-assembly by specific DNA-interactions (hybridization) were first reported in 1996 [3,4]. By modification with short synthetic DNA molecules, nanoparticles became addressable [5]. Typically, thiol-modified DNA is used for immobilization on gold nanoparticles. Depending on the size of the nanoparticles, up to several hundred DNA-molecules are coupled to one nanoparticle. The light scattering properties of metal nanoparticles are known [6] and were applied for the detection of DNA-binding by an evanescent wave [7]. Gold nanoparticles were also used as label for a microgravimetric analysis of DNA [8]. An experiment aiming at assembling colloidal gold rationally and reversibly into macroscopic aggregates (FIGURE 1A), used two batches of 13 nm particles with different noncomplementary single-stranded (ss)DNA (A´ and B´) and a connecting DNA with sticky ends (A, B) complementary to each of both ss DNA [9]. The batches of 13 nm particles exhibit a dark red color (typical for colloidal gold of this size), which changes by addition of the connecting DNA immediately to purple. It becomes clear with a pinkish-gray precipitate over the course of several hours, that points to

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+ B

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Assays based on glass slides

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Figure 1. 1 Schemes of nanoparticle-based hybridization assays for the detection of DNA. a. Homogeneous assay that applies 2 batches of nanoparticles with sequences (A’, B’) both complementary to the DNA of interest. If this DNA is present, it will cross-link the nanoparticles, resulting in a color change of the solution. b. Substrate-based assay using immobilized capture DNA with a sequence A’ that is complementary to the DNA of interest A. c. Substrate-based assay comparable to the assay described before but using a sandwich approach where the DNA of interest (A, B) is bridging a nanoparticle-labeled DNA A’ with a surface-immobilized capture DNA B’.

the formation of a DNA-colloid precipitate. This phenomenon can be explained by the creation of colloid–colloid bonds due to DNA hybridization, which finally precipitates the grown complexes. It is reversible, heating above the dissociation temperature of the connecting DNA duplexes (80°C) results in a red color of the solution, pointing to separated DNA-colloids. This DNA-related color-change was used for a colorimetric differentiation of polynucleotides (A, B) with single base imperfections; the detection could be improved by subsequent transfer of the complexes onto reverse-phase silica plates [9–11]. About 10 fmol of an oligonucleotide could be detected with this unoptimized system. Nanoparticle-labeled DNA on solid supports

Although the described colorimetric DNA-assay is quite unique regarding simplicity and sensitivity, it has the disadvantage of a homogeneous assay regarding higher parallelization. Therefore, a solid-support-based assay would be helpful, especially in combination with today’s microstructuring possibilities. The solution experiments with DNA-modified nanoparticles and complementary DNA were adapted to solid-support-based systems [12–14]. These experiments had several potential applications. One possibility is the design of interfaces by molecular-nanotechnological means. A stepwise construction of nanoparticle architectures was demonstrated [12]. In principle, one can synthetically program interparticle distances, particle periodicities and particle composition through choice of DNA sequences. Another technological application

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is envisioned by the demonstration of the control of nanoparticle deposition by support-immobilized DNA-sequences [13]. DNA–nanoparticle complexes bind preferably on locations with complementary DNA, thereby guiding the particles to surface regions modified with the corresponding DNA. This effect was already used for the positioning of larger gold structures (Au nanowires 0.2 µm diameter and up to 6 µm in length) on substrates [15].

A typical affinity-based chip assay uses complementary capture molecules microarrayed in a defined pattern on a solid support to bind the molecules of interest from the solution. The binding is detected using labeled molecules, with fluorescence readout as standard method. The described chip scheme was adapted for the use of nanoparticles as labels and applied to slides [16] and microstructured DNA chips [17]. There are two general set-ups for such assays regarding the molecule of interest (target) and the labeled molecule (probe). The first uses targets that are labeled, resulting in a direct binding as shown in FIGURE 1B. The other type is the sandwich assay, where the target is bridging the chip-immobilized capture molecule and the probe labeled with the nanoparticle (FIGURE 1C). A realization of the first (the direct) type is described in FIGURE 2. Using standard glass slides as support, an array of binding regions of 1 x 1 mm was microstructured using standard photolithographic tools [17]. Amino-modified capture DNA was covalently immobilized in these regions using a silanization procedure [14]. Different capture sequences were used for each column. The two outer columns are noncomplementary to the labeled probe; both center columns exhibit a complementary sequence. Readout was with a flatbed scanner. The scheme in FIGURE 2B describes the case of specific binding for complementary sequences of capture and labeled probe. After incubating the slide with the labeled probe (left), a DNAmediated binding of the nanoparticles to the surface occurs (center). The inset shows an array of 2 x 4 squared binding regions after specific nanoparticle binding. The pattern of the array is hardly visible judging against the right inset (which shows the same region after enhancement). Compared with the high optical contrast of high-density nanoparticle layers (FIGURE 3A), this low visibility points to a low density of surfacebound nanoparticles. To improve the contrast of the array, a silver enhancement step was applied [16], as known from electron and light microscopy [18]. This process results in a growth of the immobilized particles, as sketched in the right scheme (FIGURE 2B, a more detailed microscopical view of this process is given in FIGURE 4). Now, the optical contrast as been improved significantly, so that the squares of immobilized capture DNA are clearly visible (inset). A control experiment with noncomplementary capture DNA is described in FIGURE 2C. The binding is minimized, so that the gold particles alone are not visible at all and even the enhancement (which was the same for the arrays in FIGURE 2B & C) results in hardly visible squares. The capture DNA and also the label DNA were only applied into

Expert Rev. Mol. Diagn. 2(2), (2002)

Gold nanoparticle label

Complementary sequences (specific binding) A'

A b Noncomplementary sequences (unspecific binding) A'

c

a

B

Figure 2. Slide-based hybridization assay (30 base pairs) with nanoparticle labeling and subsequent silver enhancement. a. Overview of the slide used in the experiment after silver enhancement (standard glass slide), using a standard flatbed scanner. The binding areas (size 1 x 1 mm) are visible as 4 columns of 8 squares each in the upper part of the slides, whereby the first and fourth column exhibits only a weak signal due to noncomplementary sequences. Column 2 and 3 exhibited capture DNA (30 bases) complementary to the DNA on the nanoparticles and the resulting specific binding yields a significantly higher signal. b. Scheme for specific binding of the complementary sequences and images of selected binding areas from the complementary sequences (column 2 and 3). After nanoparticle binding the immobilized particles are enhanced by specific silver deposition. c. In the case of noncomplementary sequences, only a few nanoparticles adsorb due to unspecific binding, resulting in a weak signal even after enhancement.

the squares but the silver enhancement solution covered the whole slide. Therefore, the background signal repeating the array pattern points to an insignificant background of the silver enhancement in the surrounding areas and the signal in the squares are probably due to a minimal unspecific nanoparticle binding, which was observed between the binding squares (FIGURE 3C).

variations in structure size and pattern. A typical glass substrate with microstructured DNA-spots is shown in FIGURE 3A. It was imaged in reflection mode. Therefore, the glass background appears dark and surface areas bearing nanoparticles are bright. The smallest features of 2 µm are clearly visible in this overview micrograph, pointing to resolution better than 1 µm achievable with standard microscopical set-ups and without the need for fluorescence optics and filter sets.

Microstructured DNA-chips

The experiments described in FIGURE 2 demonstrated the specificity and the sensitivity of silver-enhanced nanoparticle detection on the slide scale using comparatively large binding areas for each spot. However, for an application on a highly-paralleled chip scale, the binding areas have to be minimized. Therefore, experiments were designed using areas with sizes reaching down to the lower micrometer range. For this size range, application of different capture DNA on each spot becomes difficult because standard spotting procedures address the medium micrometer range. To yield smaller areas of capture DNA, techniques using photosensitive protection groups (which are activated by standard microlithographic mask exposure or a digital mirror-controlled laser) and a wet-masking technique were proposed [19–21]. We used for our experiments microfabricated substrates that were subjected to an ‘on-chip’ DNA synthesis [17]. Although this approach is limited to only one sequence per chip, it is open to

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Single-particle detection

Although an optical readout is useful as an established and potentially highly-paralleled method, a high-resolution imaging method is required for the establishment of the technology. Important parameters are, e.g., the density of nanoparticles bound specifically compared with background binding or the relationship between solution concentration and the density of immobilized particles. To access this information, a method with the potential of single-label resolution is optimal. The individual label is easily visualized using scanning force microscopy (SFM) – also known as atomic force microscopy (AFM) – an imaging tool optimal for characterization of thin layers and structures with nanometer resolution [22]. The SFM image of a part of the structure in FIGURE 3A is shown in FIGURE 3B. The height is brightness-coded, so bright structures are high. A more detailed view of a nanoparticle-

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labeled surface is shown in FIGURE 3C. Now the individual nanoparticles can be easily distinguished (arrows) in regions with lower nanoparticle density between the spots in the lower left and upper right corner. The simple visualization of individual labels is an important advantage for the development of a new labeling scheme because it allows an easy access of important parameters such as signal or background level, degree of unspecific binding etc. Counting the nanoparticles in an area of known size allows for a straightforward classification, which was, e.g., used for the determination of the signal and the background level of nanoparticle-labeled microstructured DNA spots for optical detection [17]. Another application is the investigation of the unspecific binding of DNA-modified nanoparticles on gold surfaces modified with noncomplementary DNA and in an unmodified state [23]. Beside the visualization of the particles, scanning force microscopy is very useful for the characterization of the surface roughness of the binding spots. Therefore, it was used for a study of surface roughness of silane surfaces, which can be very smooth, down to subnanometer surface roughness over a scale of several micrometers [24]. Novel optical readout devices

The image in FIGURE 3A demonstrates that surfaces covered with colloidal gold particles exhibit a strong optical signal. In the case of higher particle densities and micrometer areas they are visible by the naked eye (e.g., 50 µm squares as demonstrated in [17]). The presence of the particles is detectable not only by their surface plasmon resonance in a dark-field microscopical set-up [25] but also by simple transmission or reflection. Due to the metallic character of the particles, a significant reflection occurs already at small spots, as it was demonstrated with 4 µm structures and a millisecond exposure time [26]. These studies were made using an optical microscope but the observed high contrast let one consider also less sophisticated devices, which is especially interesting

A

B

for an envisioned broad distribution of future diagnostic devices. Assuming sufficient contrast, the next important point would be the resolution. Ongoing discussions in the microarray community point to at least 9 pixels (3 x 3) per spot as a minimum. Assuming a 200 µm spot size as minimum for today’s spotting technologies, 1 pixel should represent less than 70 µm. Another point is the preference for a method requiring only one exposure compared to scanning techniques. Based on an array of 10 x 10 spots (separated by 200 µm), imaging devices should exhibit a minimal pixel number of about 60 in both directions. This number could be already fulfilled by the simplest available webcam. However, one would need an optical adoption to project the chip onto the CCD at a 1:1 scale. This problem is already solved in optical scanners, which are (especially in their flatbed models) widely distributed. A resolution of 70 or better 20 µm per pixel is equivalent to 340 (1200) dpi, respectively, which is standard in today’s low-cost models. Another point is the number of gray levels required. Since the detection scheme is still in development, it is not yet at the limits of gray level resolution. For the start an 8-bit approach is probably a reasonable value, which should be verified in the course of further developments of the method. A first confirmation of this approach used a flatbed scanner for the detection of silverenhanced nanoparticle-labeled spots on a glass slide [16]. Other experiments demonstrated the application of scanners for nanoparticle labels even without enhancement [27]. Summary & conclusion

Gold nanoparticles are a promising novel label for microarray and chip technology. Especially the high stability and the option of robust and simple detection point to an advantage over the standard fluorescence labels. Although commercial applications still have to be developed, a variety of proof-ofprinciple experiments in the research laboratories point to a powerful technology for molecular diagnostics.

C

Figure 3. Micrographs of a microstructured substrate containing binding areas with capture DNA (20 bases) after nanoparticle labeling. labeling. a. Optical contrast (reflection). The test pattern featuring various structure sizes is clearly visible in the optical contrast due to the reflection of the areas containing nanoparticles. b., b. c. Scanning force micrographs of a sample similar to a., a. showing the pattern created by areas with a high density of the bright nanoparticles, which are resolved as individual particles in c. Height is brightness-coded and higher structures are brighter. The brightness difference between the lightest parts of the particles and the background gray value corresponds to a height of 30 nm.

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Gold nanoparticle label

A

B

C

Figure 4. Metal enhancement of immobilized nanoparticles. DNA-modified nanoparticles were specifically bound to surfaces with immobilized complementary capture DNA using linker DNA (96 bases, 30 bases overlap to each label and capture DNA) and subjected to a metal enhancement for 4 (A), (A) 6 b. and 8 c. min, respectively.

Expert opinion

DNA chip diagnostics will expand when critical issues, especially with regard to reproducibility and standardization, can be solved. These problems are closely related to the used fluorescence labels, so every complementary marker should be welcome. Gold nanoparticles show a promising potential for higher stability, so they could be the method of choice for further development in optical chip technology. If these problems are solved, a higher parallelization of diagnostical assays will be observed with the expansion of chipbased diagnostics. The goal is the development of low-cost methods on one hand for individual test or test family but on the other hand to provide more diagnostic information. Five-year view

For a more widespread use of chip diagnostics, the chip assays have to be standardized, so that new applications should be realized simply by adaptation of standard assays. If this point can be reached, further progress will be of tremendous speed. However, for achieving this goal, the signal in chip assays has to be robust and well-characterized, especially regarding response to DNA presence and concentration. From today’s perspective, gold nanoparticles are a system for robust signals, already confirmed by first proof-of-principle experiments. It is likely that this labeling technique will accomplish to become second to or maybe even to become equivalent to fluorescence labeling. Fluorescence has – beside the long tradition and therefore experiences – advantages compared with nanoparticles, namely the potential for multiple labels and the small size. Therefore, it is to expect that each applications will have its own set of requirements, which will lead to parallel use of fluorescence and novel markers like gold nanoparticle. A fully new field of application for nanoparticle labels is opened by the proposal of an electrical detection, which was

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experimentally verified recently [24]. For this purpose, capture DNA is immobilized in the gap of microstructured electrodes and binds nanoparticle-labeled DNA. Binding events are detected by silver-enhancement – that results in a conductive layer spanning the gap – prior to resistance measurements. Therefore, the whole optical set-up required today for nanoparticle detection would become obsolete, replaced by electrical circuits which are inexpensive and are easily integrated into small and compact readers – with a potentially large impact for diagnostical devices. Acknowledgements

• We would like to acknowledge the initial contribution of J Köhler for the establishment of this work, H Saluz for helpful discussions and advice, G Maubach, I Frank, A Steinbrück, W Straube, J Reichert, E Birch-Hirschfeld, M Sossna, F Jahn, H Porwol and K Kandera for help and technical assistance during the experiments. • The VCI/BMBF (Liebig scholarship to W.F.) and the DFG (Fr 1348/1–4) are acknowledged for funding this work. Key issues • Chip-based hybridization enables highly-paralleled DNA detection with minimal sample volume. • Today’s standard label for DNA chip detection is fluorescence, with disadvantages regarding dye stability and required equipment for readout. • Gold nanoparticles are potentially stabile and easily detectable. • Proof-of-principle experiments demonstrated the successful use of nanoparticles for DNA detection.

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RL. Homogeneous, nanoparticle-based quantitative colorimetric detection of oligonucleotides. J. Am. Chem. Soc. 122, 3795–3796 (2000).

References

Papers of special note have been highlighted as: • of interest •• of considerable interest Hayat MH. Principles and Techniques of 1 Electron Microscopy. Biological Applications, Hayat MH (Ed.) Van Nostrand Reinhold, New York, NY, USA (1970). 2

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Alivisatos AP, Johnsson KP, Peng X et al. Organization of 'nanocrystal molecules' using DNA. Nature 382, 609–611 (1996). •• The start of the new field of DNAnanoparticle complexes. Mirkin CA, Letsinger RL, Mucic RC, 4 Storhoff JJ. A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature 382(6592), 607–609 (1996). •• See reference [3].

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Storhoff JJ, Elghanian R, Mucic RC, Mikron CA, Letsinger RL. One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J. Am. Chem. Soc. 120, 1959–1964 (1998). Interesting DNA-test based on colorchanges. Taton AT, Mucic RC, Mirkin CA, Letsinger RL. The DNA-mediated formation of supramolecular mono-and multilayered nanoparticle structures. J. Am. Chem. Soc. 122(26), 6305–6306 (2000). Niemeyer CM, Ceyhan B, Gao S, Chi L, Peschel S, Simon U. Site-selective immobilization of gold nanoparticles funtionalized with DNA oligomers. Colloid Polymers Sciences 279, 68–72 (2001). Möller R, Csáki A, Köhler JM, Fritzsche W. DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold. Nucleic Acids Research 28(20), e91 (2000). Mbindyo JKN, Reiss BD, Martin BR, Ketaing CD, Natan MJ, Mallouk TE. DNA-directed assembly of gold nanowires on complementary surfaces. Adv. Materials 13(4), 249–254 (2001). Taton TA, Mirkin CA, Letsinger RL. Scanometric DNA array detection with nanoparticle probes. Science 289(5485), 1757–1760 (2000). DNA test based on enhanced nanoparticles using a flatbed scanner for detection. Reichert J, Csáki A, Köhler JM, Fritzsche W. Chip-based optical detection of DNAhybridization by means of nanobead labeling. Anal. Chem. 72(24), 6025–6029 (2000).

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Hacker GW, Silver-enhanced colloidal gold for light microscopy. In: Colloidal gold: principles, methods, and applications. Hayat MA (Ed.) Academic Press. 297–321 (1989).

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Fodor SPA, Rava RP, Huang XC, Pease AC, Holmes CP, Adams CL. Multiplexed biochemical assays with biological chips. Nature 264 555–556 (1993).

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Singh-Gasson S, Green RD, Yue Y et al. Maskless fabrication of light-directed oligonucleotide microarrays using a digital

micromirror array. Nature Biotechnol. 17(10), 974–978 (1999). 21

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Fritzsche W. Scanning force microscopy: a microstructured device for imaging, probing, and manipulation of biomolecules at the nanometer scale. In: Microsystem technology: a powerful tool for biomolecular studies. Köhler JM, Mejevaia T, Saluz HP (Ed.), Birkhäuser, Basel, Switzerland, 353–370 (1999).

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Csáki A, Möller R, Straube W, Köhler JM, Fritzsche W. DNA monolayer on gold substrates characterized by nanoparticle labeling and scanning force microscopy. 29, e81 (2001).

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Möller R, Csáki A, Köhler JM, Fritzsche W. Electrical classification of the concentration of bioconjugated metal colloids after surface adsorption and silver enhancement. Langmuir 17 5426–5430 (2001). First demonstration towards an electrical detection of metal nanoparticles based on enhancement.

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Schultz S, Smith DR, Mock JJ, Schultz DA. Single-target molecule detection with multicolor optical immunolabels. Proc. Natl Acad. Sci. USA 97, 996–1001 (2000).

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Köhler JM, Csáki A, Reichert J, Möller R, Straube W, Fritzsche W. Selective labeling of oligonucleotide monolayers by metallic nanobeads for fast optical readout of DNAchips. Sensors and Actuators 76(B), 166– 172 (2001).

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Fritzsche W, Csaki A, Möller R. Nanoparticle-based optical detection of molecular interactions for DNA-chip technology. SPIE 4626 (2002) (In Press).

Affiliations • Andrea Csáki • Robert Möller • Wolfgang Fritzsche Molecular Nanotechnology Group, Institute for Physical High Technology PO Box 100239, 07702 Jena, Germany Tel.: +49 364 120 6304 Fax: +49 364 120 6344 [email protected]

Expert Rev. Mol. Diagn. 2(2), (2002)