IOP PUBLISHING
JOURNAL OF PHYSICS D: APPLIED PHYSICS
J. Phys. D: Appl. Phys. 42 (2009) 055507 (7pp)
doi:10.1088/0022-3727/42/5/055507
Holographically encoded microparticles for bead-based assays Sam W Birtwell1,4 , Shahanara Banu2 , Nikolay I Zheludev3 and Hywel Morgan2 1
School of Physics and Astronomy, University of Southampton, Highfield, Southampton, SO17 1BJ, UK School of Electronics and Computer Science, University of Southampton, Highfield, Southampton, SO17 1BJ, UK 3 Optoelectronics Research Centre, University of Southampton, Highfield, Southampton, SO17 1BJ, UK 2
E-mail:
[email protected]
Received 11 September 2008, in final form 12 January 2009 Published 19 February 2009 Online at stacks.iop.org/JPhysD/42/055507 Abstract We demonstrate a re-writable, high capacity holographic encoding technique for multiplexed bead-based suspension assays. The microparticles are made from SU8 doped with a photochromic diarylethene dye and manufactured using multilayer photolithography and dry etching. Each particle is encoded with a unique hologram, whose diffraction pattern consists of bright and dark regions, representing a binary number that identifies the particle. Theoretically up to 1024 unique codes are available on a 100 µm particle using this method, when the code is read with a standard 2/3 CMOS camera. Encoding capacities of 512 unique codes have been demonstrated on a 500 µm SU8 particle. The code is thermally stable for 3 days at 25 ◦ C, and once written, the code can be erased and re-written once whilst still remaining readable. The code can be written into the particle during an assay experiment (no pre-encoding is required) and requires simple optics for reading.
is one of the most common techniques used to indicate that a chemical reaction has taken place on a bead. Graphical methods, which depend on visual recognition of a spatial code pattern, whilst generally requiring larger particles, have been shown to have a capacity of the order of 106 unique codes [5]. Recently, we reported a new approach to bar-coding based on patterning particles with micrometre-sized diffraction gratings. The particle is identified from the unique distribution of light diffracted from the multiple superimposed gratings [7–9]. Here we report on the development of a new diffractive microparticle encoding technique and encoding medium that uses holograms written into photosensitive microparticles. The technique allows encoding a particle with a diffraction pattern that meets the requirements of binary storage and recovery of information (including error analysis). In addition, the technique is shown to have a key advantage over all existing microparticle encoding methods, in that the data encoded in the particle can be erased and then re-written, which could be useful in many areas, not least split-and-mix combinatorial synthesis. Encoded holograms have been widely researched for high capacity computer data storage [10]. In this case, ‘pages’
1. Introduction Recent advances in the areas of genomics, proteomics, highthroughput screening and combinatorial chemistry have led to the development of techniques for multiplexed chemical analysis. One such technique is bead-based analysis, which offers a number of advantages over conventional array-based approaches. Bead-based analysis uses solid micrometresized particles as platforms for chemical or biochemical reactions or assays. In order to simultaneously perform more than one assay, each particle must carry a unique identifier indicative of the molecule on its surface, i.e. a barcode. A number of approaches have been developed for bar-coding particles [1], the most promising of which are based on spectral [2–4] or graphical [5, 6] encoding. Fabricating particles containing several different fluorescent quantum dots with discrete emission intensities, it is possible to produce several hundred unique codes [4]. However, encoding with fluorescence has many problems; not least that fluorescence 4
Author to whom any correspondence should be addressed. Current address: The School of Electronics and Computer Science, University of Southampton, Highfield, Southampton, SO17 1BJ, UK. 0022-3727/09/055507+07$30.00
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of binary data are stored as holograms in a photosensitive medium. Each page is an array of pixels that is either bright or dark, representing a zero or one. Encoding of microparticles for assay applications places somewhat different requirements on the photosensitive medium to that used for computer data storage. For bead-based assays the particle must be made from a material which is non-toxic and easily formed into well-defined shapes. Furthermore, it should be amenable to subsequent immobilization of molecules. We use the novolacepoxy resin SU8 (a negative resist), which is widely used for microfabrication and micropatterning [11–13] and as a support material for the direct attachment of biomolecules [14–18]. We have previously used SU8 to manufacture micronmetresized bars (i.e. beads) encoded with diffraction gratings and demonstrated multiplexed assays [19]. The particles have also been used in conventional multistep solid-state synthesis of oligonucleotides and peptides [20]. In order to holographically encode SU8 particles in a re-writeable manner, the SU8 is doped with a photosensitive material. Many organic molecules have been proposed for the creation of re-writable photosensitive polymers. The most thermally stable of these are the diarylethene-type dyes [21–24], one of which was used in this work. We describe the encoding of dye-doped SU8 with holograms of spot-arrays that represent a binary number which uniquely identifies each particle. The dye is shown to have a thermal decay lifetime of a few days in SU8 and to form high quality binary page holograms in particles, with excellent binary signal-to-noise (SNR) ratio. We have demonstrated that the holograms can be written after manufacture and detaching of the particles from the substrate (lift-off), and thus writing of the codes can potentially be achieved on-chip in a microfluidic assay analysis system.
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Figure 1. The diffraction pattern used to encode microparticles is a series of bright and dark regions. (a) Two orthogonal lines of bright spots define a code grid (grey dashed lines), upon which spots are placed to represent a ‘1’ bit. Grid spaces left blank indicate a ‘0’ bit. (b) An example depicting the code 0110 1011 1111 1001. (c) The theoretical encoding capacity of different hologram sizes, for various values of lens focal length f , when the reconstructed code is observed on a standard 2/3 camera sensor. The values of f (in millimetres) are given above each curve. The bits reconstructed from each size of hologram are spaced by a distance a, which gives the required Fourier transform size. The largest rectangular array of bits spaced by a that fit into the sensor area was calculated and the reference row and column of bits removed. The remaining n bits are available for encoding; the number of unique binary codes available using these bits is 2n .
2. Encoding concept The diffraction pattern from the hologram written into a particle is the code that identifies that particular particle. An example of such a code is shown in figures 1(a) and (b). The pattern consists of two sections. The first section is present in all patterns and comprises two orthogonal lines of bright spots, defining a ‘code grid’, see figure 1(a). The code that identifies a particular particle is written onto this grid by either placing a bright spot at a bit position (corresponding to a‘1’ bit), or leaving the bit position blank (‘0’ bit). As an example, the 16-bit code 0110 1011 1111 1001 is shown in figure 1(b). A hologram that produces a particular diffraction pattern (when read) is written into the photochromic SU8 using a standard Fourier holography method. A spatial light modulator (SLM) is programmed to produce a pattern of reflected light corresponding to the code that the hologram should produce when read. A laser beam reflected from the SLM (the ‘signal’ beam) is focused onto the photochromic SU8 by a lens of focal length f , forming the Fourier transform of the code pattern on the SU8 surface. This beam is interfered with a reference beam, creating the hologram in the photochromic material. When the hologram is illuminated by a readout beam similar to the original reference beam, a pattern of light is produced
which is focused by another lens with the same focal length f , creating a reconstruction of the code pattern, which is imaged on a camera on the reverse side of the substrate. The encoding capacity of this hologram depends on the bit spacing a in the code pattern, and the size of the observable image space (which determines the number of spots of spacing a that can be used for encoding). The observable image space is fundamentally limited by the size of the sensor in the camera used to observe the reconstructed code hologram. The bit spacing is set by the parameters of the lens used during the writing process and the required hologram size. This is because the hologram size is approximately equal to the distance between the first order peaks of the Fourier transform of the code pattern, scaled by the lens focal length, f . This distance is equal to λf/a, where λ is the wavelength used for writing. The encoding capacity of different hologram sizes was calculated for a number of values of f , with a standard 2/3 (active area 8.5 × 6.8 mm2 ) camera sensor (figure 1(c)). Theoretically, up to 1024 codes are available on a 100 µm particle with f = 50 mm. For a 2
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interval, to minimize sample heating. After exposure, the SU8 was baked on a hotplate (temperature ramp from 50 to 95 ◦ C at 2 ◦ C min−1 ) and left to cool to room temperature on the hotplate. To produce high contrast holograms, multiple layers of SU8 were deposited on top of each other (figure 3(a), step 3), using the same procedure. A four-layer structure (figure 3(a), step 4) produced acceptable hologram quality. The particles were manufactured by dry-etching flat plates of the material (figure 3(a)). A 50 nm aluminium release layer was evaporated onto a glass wafer and then, following (steps 1–4), a four-layer flat plate of SU8 was manufactured. For dry etching a Ni mask was used. First a chrome/gold layer is evaporated onto the SU8 (step 5) followed by 2.8 µm of S1828 (positive photoresist), soft baked at 95 ◦ C for 35 min (step 6). Figure 2. Absorption spectra for the open-ring (dashed line) and This layer was patterned to define the particles. Exposed closed-ring (solid line) forms of the photochromic diarylethene dye resist was developed with MF319 solvent (Microchem Corp.) 1,2-bis(2,4,5-trimethyl-3-thienyl)-cis-1,2-dicyanoethene dissolved in SU8. The structures of the two forms are shown (inset), together leaving holes that define the particles. Nickel was electroplated with typical wavelengths that cause the photochemical into the holes (step 9) by electroplating in a Watt’s bath [26] transformations between the two forms. containing an aqueous solution of nickel sulfate (NiSO4 · 7H2 O, concentration 267 g L−1 ), nickel chloride (NiCl2 · hologram with this encoding capacity, there are 80 code bits in 6H O, concentration 100 g L−1 ) and boric acid (H BO , 2 3 3 the diffraction pattern reconstructed from the hologram; non- concentration 35 g L−1 ). The final nickel thickness was 5 µm. erasable holographic mass data-storage systems using different The resist was stripped and exposed gold/chrome removed by photopolymers have been reported with around 29 000 bits in ion-beam milling (Oxford Instruments Ionfab 300 plus), using the reconstructed hologram [25]. argon ions (flow rate of 6 cm3 min−1 ). The exposed SU8 was dry-etched in an Oxford Plus Plasma etcher (gas flow rates: 60 cm3 min−1 O2 , 3 cm3 min−1 SF6 , forward power = 200 W, 3. Particle fabrication reflected power = 0, chamber pressure = 150 mT, plasma To manufacture photosensitive SU8 particles, the SU8 was table at 10 ◦ C, etch time = 45 min [27]) and the nickel doped with the photochromic diarylethene dye 1,2-bis(2,4,5- removed with 30% aqueous FeCl3 (70 ◦ C for 30 s) and the trimethyl-3-thienyl)-cis-1,2-dicyanoethene (TCI-Europe N.V.), gold/chrome layer with gold etchant (I2 and KI) and chrome which has previously been characterized in matrices of the etchant (acetic acid and ceric ammonium nitrate). The particles polymers PMMA and PVA [22]. This dye was chosen for were removed from the wafer by sonicating in aluminium etch its high solubility in the solvents used for SU8; gamma- (MF319, Microchem Corp.) diluted with water. The particles butyrolactone or cyclopentanone. The two photochemical were pipetted onto a microscope slide and left to dry in air, reactions of the dye molecule (dissolved in SU8) in the open- before being protected with a cover slip and used for hologram ring form are a conrotatory cyclization (figure 2, inset), or a writing. SEM images of the 500 × 1500 µm2 particles are shown cis-trans isomerization [23]. During the cyclization reaction, in figure 3(b). Un-patterned sheets of dye-doped SU8 were the molecule’s absorption spectrum changes so that an absorpalso used, to characterize holograms formed in the material. tion band 500–600 nm wavelength region appears, as shown in figure 2. Illumination of the closed-ring form with longer wavelength light (500–600 nm) returns the dye to the open-ring 4. Hologram recording and readout form. This reaction was used for recording the holograms. SU8 particles were fabricated using photolithography To prepare for writing, a sample of photochromic SU8 was and reactive ion-etching, following the process outlined in first exposed to a broadband UV source (20 mW cm−2 centred figure 3(a). 5% wt dye was mixed with SU8-2015 (in at 365 nm) for 30 min which leads to switching of the dye cyclopentanone, Chestech Ltd.) and spun onto a 0.7 mm thick into the closed-ring form. Holograms were written into the boro-silicate float glass substrate (Schott AG), pre-treated with photochromic dye-doped SU8 sheets using the setup shown in Ti-prime adhesion promoter (Microchem Corp.) at 8000 rpm the schematic in figure 4. The 532 nm beam from a frequency to produce a layer ∼8 µm thick (figure 3(a), step 1). This doubled Nd : YVO4 laser is split into two. One beam is was baked for 1 min at 65 ◦ C, then 3 min at 95 ◦ C, and left to expanded by 10 times (L1 and L2, incorporating pinhole P relax for 2 h at room temperature. The sample was exposed for spatial filtering) and reflected from a Texas Instruments to UV light (365 nm, intensity 10.5–11.0 mW cm−2 ) using an DLP® SLM, which reflects light only from the required code EVG620 mask aligner (figure 3(a), step 2). An exposure points towards the SU8 substrate. A lens of focal length f time of 8 min was required to fully cross-link the SU8 due (L3) forms the Fourier transform (FT) of the code pattern to UV absorption by the dye (compared with a typical time on the SU8 surface. The spacing between the bits in the of approximately 12 s). The exposure was performed in 48 holograms was 220 µm, which together with the focal length intervals of 10 s each, with a dark time of 20 s after each f = 50 mm resulted in a calculated FT size of 400 µm. The FT 3
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Figure 3. (a) Schematic of the process used to manufacture the photochromic dye-doped SU8 microparticles. (b) SEM images of 500 × 1500 µm2 microparticles manufactured from SU8 doped with this dye.
Figure 4. Schematic diagram of the setup used to write holograms into photochromic dye-doped SU8. BS: beamsplitter, L: lens, M: mirror, P: spatial filter. Inset shows coordinate definitions for sample rotation (z-axis into plane of page).
the pattern in figure 5(a) is shown in figure 5(b). This image shows that the pattern has been successfully reproduced with minimal distortion. Cross sections through the image (figures 5(c) and (d)) demonstrate high contrast between bright and dark regions of the encoded diffraction pattern from the hologram. The mean SNR of 1 bit intensity to 0 bit intensity for 50 holograms is 5.9 ± 1.2. The holograms are decoded using a dedicated computer algorithm. The algorithm uses a Gaussian filter to remove noise caused by scattered light, followed by binarization of the image into black and white. A ‘region finding’ method [28] is then used to identify and label each white area in the image. The centre of each white area is found, and from these a set of coordinates for each code spot in the image is produced. The frame spots (highlighted grey in figure 5(a)) are found and a ‘grid’ of expected spot positions constructed, as in figure 1(a). Each of these expected positions is then checked for in the image code spot coordinate list: if a coordinate is present, the position is assigned a ‘1’ bit, otherwise a ‘0’ bit is assigned. The resulting string of bits constitutes the code on the hologram. The Gaussian filter used for noise
of the code pattern is interfered with an un-patterned reference beam from the same 532 nm laser. The resulting interference pattern is recorded in the photochromic material, forming the hologram. The diffraction pattern is reconstructed from the hologram by illumination with a 633 nm HeNe laser, and focussed by a second lens (L4) of focal length f = 50 mm onto a CMOS camera sensor (1280×1024 pixels, Prosilica EC1280, integration time 41 ms). A similar process was carried out to record holograms into the individual particles, with a code bit spacing of 490 µm. An example of a code patterned onto the signal beam using the SLM is shown in figure 4(a). White spots represent points at which light is reflected towards the sample by the SLM; the rest of the beam is reflected away from the sample. In this example the binary code string is 01000011, 11111111, 11111111, 11111111, 11011111, 11111111, 11111111, 11111111. After writing, the hologram is reconstructed by illuminating with a 633 nm laser beam. To read the code the resulting diffraction pattern is imaged on a CMOS camera. An example diffraction pattern reconstructed from a hologram written into a sheet of dye-doped SU8 using 4
J. Phys. D: Appl. Phys. 42 (2009) 055507
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Figure 5. (a) An example of a code pattern written onto the signal beam using a SLM, indicating the grid-defining spots (grey shading). (b) An example image of a code reconstructed from a hologram written into an SU8 sheet. (c) A cross-section through a row of entirely 1 bits of the hologram in (b). Comparing this cross-section with one taken through a row containing a mixture of 0 and 1 bits. (d) Shows the SNR between 0 and 1 bits. (e) An image of the reconstructed diffraction pattern from a hologram written into a 1500 × 500 × 35 µm3 SU8 particle. Image is contrast enhanced for clarity. (f ) A cross section through the un-enhanced image showing the pattern SNR.
reduction removes some code spots for holograms with SNR less than 1.8. Such low-SNR images are therefore flagged as unreliable. A trial of 100 holograms with the algorithm gave a correct reading rate of 98%, with the remaining 2% of holograms flagged as unreliable. For more robust error detection and correction during reading, one row and one column of the hologram could be allocated to parity check bits. After demonstrating the concept on SU8 sheets, holograms were written into individual SU8 particles of dimensions 1500 µm × 500 µm × 35µm, manufactured on aluminium coated glass wafers, lifted off and deposited onto glass microscope slides. A 4 × 4 array of dots was written and an example of a reconstructed hologram from a single particle is shown in figure 5(e), together with a cross section, figure 5(f ). The mean SNR of each bit over the pattern (± standard deviation) is 1.9 ± 0.3 for a typical particle. For biochemical assays, the hologram must remain readable throughout the duration of an experiment. The openring form of the photochromic dye can decay to the closed-ring form by thermal relaxation [24], erasing the hologram. The thermal decay of the dye was characterized by reconstructing a hologram from a sheet of dye-doped SU8 at intervals (exposure to light for 1 s each time) over 4 days, storing the material in the dark at room temperature. The change in SNR with time is shown in figure 6(a), indicating that the SNR reduces by approximately 60% after 70 h (2.9 days) to a value of 2.3 and is therefore still readable after 3 days storage at room temperature.
During an assay, holograms are written into the particles, after which the particles are removed from the substrate and suspended in the reaction medium. After completion of any reaction, the particles are placed back onto the substrate for readout. The particles will obviously not return to exactly the same orientation as during writing, and the effect of particle rotation and tilt on the reconstructed code images has to be considered. The effect of particle rotation about the z-axis (see figure 4 inset for axes definitions) can be estimated by calculating the magnitude of the diffracted field from the hologram at different readout beam incident angles using the Kirchhoff diffraction equations [29]. The resulting dependence of the field strength on the readout angle depends upon many parameters: the angles of the signal and reference beams to the recording medium, the thickness of the medium and the writing and readout wavelengths. For the values of these parameters used in the hologram writing, an angular tilt of more than ±3◦ completely extinguishes the diffracted light. Allowing the particles to settle on a flat glass substrate (by gravity) keeps particle tilt well within ±1◦ , retaining >80% of the maximum image intensity. Rotation of the particle about the x-axis simply moves the reconstructed image, the only constraint being when the image no longer falls on CMOS sensor surface. For the examples shown above this limit is approximately ±5◦ . Rotational re-alignment could be accomplished in a number of ways, e.g. using dielectrophoresis of non-symmetrical particles [30] or mechanical confinement of the particles in a pitted substrate [31], or use of magnetic fields. Movement or distortion of the reconstructed image due 5
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fatigue mechanism is a cis-trans isomerization of the open-ring form of the dye to 1,2-bis(2,4,5-trimethyl-3-thienyl)-trans-1,2dicyanoethene, in accordance with previous observations [23]. Only the cis isomer can undergo the cyclization necessary for the absorption spectrum change, which allows holograms to be written into the material: a proportion of the dye transforming to the trans isomer will therefore reduce the diffraction efficiency of the resulting holograms. The isomerization is reversible, with the trans form relaxing thermally to the cis isomer over 48 h, restoring the hologram intensity to 70% of the initial value. However, further investigation will have to be performed to discover whether this is actually the case. (a)
5. Conclusions In conclusion, we have presented a new method of encoding polymer microparticles for use in bead-based multiplexed assays. The method is based on writing holograms into the particles, producing code images upon illumination. The particles are manufactured by a dry-etch process from the epoxy photoresist SU8, to which biochemical molecules can be attached. It has been demonstrated that the inclusion of a photochromic diarylethene dye into the SU8 allows encoded holograms to be written into the material. The holograms written into a flat SU8 plate have a mean SNR of 5.9 ± 1.2 on the initial write and remain stable for more than 3 days at room temperature, allowing ample time for biological experiments to be performed. The holograms can be read by a dedicated computer decoding algorithm and can be re-written once within a short time, whilst still remaining readable by the decoding algorithm. The holograms can be written into SU8 particles but with a lower mean SNR of 1.9 ± 0.3. Improving the surface quality of the particles should lead to an increase in SNR to values comparable with those obtained on the flat SU8 plates. 64-bit holograms have been successfully demonstrated on flat SU8 plates, leading to a possible encoding capacity of 1019 unique codes; 9-bit holograms have been demonstrated on 500 µm particles, giving an encoding capacity of 512 codes. Since the codes are written into the particles after lift-off from the wafer, the particles can be encoded during an experiment (i.e. the codes can be tailored to the particular assay being performed). Additionally, reading is accomplished with very simple optics, as the code is projected from the particle, requiring only a single lens to image it onto a camera sensor. The coded particles can be functionalized for the attachment of biomolecules using techniques developed previously [19]. It is highly unlikely that the attachment of biomolecules will affect the images reconstructed from the particles because of the small typical thickness of any biomolecular layer (around 5nm) and small effective refractive index difference in the layer, compared with the bulk medium surrounding the particle (typically n ≈ 0.05) [32]. Further work will concentrate on improving the SNR of the holograms on particles and fabricating smaller encoded particles with larger encoding capacities. In addition, other diarylethene dye types could lead to improvements in the number of re-write cycles and longer thermal decay times. Finally, methods for reading and writing the codes within a microfluidic system are being developed.
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Figure 6. (a) The SNR for hologram that has been kept in the dark (at room temperature) as a function of time. (b) the SNR of a hologram for increasing number of write/erase cycles. ‘1’ corresponds to the first write with no erasure. Writes 1–3 were performed within 3 h of each other. The sample was left for 48 h and then writes 4–6 performed within 5 h of each other. The data show that partial recovery of the fatigued dye occurs over time.
to tilt and rotation does not affect the readability of the code because of the frame spots which define the hologram edge. For certain applications such as combinatorial split-andmix synthesis, it would be advantageous to be able to erase and re-write the holograms encoding the particles. Diarylethene dyes have previously been shown to be re-writable [22]; when in the closed-ring form the dye can be returned to the open-ring form, and back again repeatably. In order to test the rewritability of holographically encoded SU8, 10 holograms were written into a sheet of dye-doped SU8. After recording the reconstructed images, each hologram was erased by illumination with 532 nm light. The sample was then re-illuminated with UV light to return the dye to the closedring form and the same holograms written again. This process was repeated a number of times, and the resulting SNR for each write is presented in figure 6(b). These results show that two successive different holograms can be written into the SU8 before chemical fatigue causes the SNR to fall below 1.8, rendering it unreadable. Leaving the hologram in the dark for 48 h at room temperature restores 70% of the 1st hologram SNR, allowing the material to be written two more times to produce readable holograms. This partial restoration of the hologram intensity suggests that at least part of the 6
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Acknowledgments The authors would like to thank Katie Chamberlain for manufacturing assistance, Jacek Lapinski for assistance with the electroplating of the dry-etch masks, Peter Roach and Rohan Ranasinghe for helpful discussions on the dye chemistry and the EPSRC and the Basic Technology Program of the Research Councils UK for funding.
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