David M.
Love
*,
Kunal N.
Vyas
,
Amalio
Fernández-Pacheco
,
Justin
Llandro
,
Justin J.
Palfreyman
,
Thanos
Mitrelias
and
Crispin H. W.
Barnes
Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE, UK. E-mail: david.michael.love@gmail.com
First published on 7th January 2015
We present a new composite element (CE) bit design for the magnetic bit encoding of suspended microcarriers, which has significant implications for library generation applications based on microfluidic combinatorial chemistry. The CE bit design consists of high aspect ratio strips with appropriate dipolar interactions that enable a large coercivity range and the formation of up to 14 individually addressable bits (16384 codes) with high encoding reliability. We investigate Ni80Fe20 and Co CEs, which produce coercivity ranges of 8–290 Oe and 75–172 Oe, respectively, showing significant improvements to previously proposed bit designs. By maintaining the total magnetic volume for each CE bit, the barcode design enables a consistent stray field for in-flow magnetic read-out. The CE bit design is characterised using magneto-optic Kerr effect (MOKE) measurements and the reliability of the design is demonstrated in a multi-bit encoding process capable of identifying each bit transition for every applied magnetic field pulse. By constraining each magnetic bit to have a unique switching field using the CE design, we enable sequential encoding of the barcode using external magnetic field pulses. We therefore discuss how the new CE barcode design makes magnetically encoded microcarriers more relevant for rapid and non-invasive detection, identification and sorting of compounds in biomolecular libraries, where each microcarrier is for example capable of recording its reaction history in daisy-chained microfluidic split-and-mix processes.
Previously, magnetic encoding of microcarriers was proposed for multiplexed point-of-care diagnostic applications, where nominally identical tags are encoded using coercivity tuned magnetic bits by externally applied magnetic field pulses,6,7,9–11 offering unique advantages in mass fabrication and scaling. The idea formed an unconventional approach12 to other suspension assay technologies, where carrier detection was typically based on nano-particle binding,13 optical barcodes14–16 or fluorescence.17,18 Coercivity tuning on the other hand, is achieved by patterning magnetic strips with different aspect ratios (Fig. 1a), giving each bit a unique field amplitude at which its magnetisation reversal occurs (i.e. switching between a 0 and 1 state) and allowing identical microcarriers to be uniquely encoded with a multi-bit code. It has been shown that batches of such encoded microcarriers can be functionalised with two different probe molecules on two distinct surfaces,19,20 using the incorporated thiol and epoxy surface groups. By also including control molecules on each microcarrier, a bioassay is capable of identifying true positives/negatives by probing the assay conditions using control targets purposely included in the analyte. When fluorescence of the target molecules indicates positive binding, the magnetically encoded microcarriers can be identified by the magnetic stray fields of their unique barcodes in-flow, as has previously been demonstrated8 using an integrated tunnelling magnetoresistance (TMR) sensor (illustrated in Fig. 1b).
Fig. 1 (a) Illustration of the coercivity (Hc) tuning technique and the original barcode design,6,7 where the coercivity distribution of ‘bits’ is determined by varying the aspect ratios of the ferromagnetic elements. (b) Illustration of the use of magnetically encoded microcarriers, with fluorescence detection and in-flow magnetic identification as previously demonstrated8 using polydimethylsiloxane (PDMS) moulded microfluidics with an integrated tunnelling magnetoresistance (TMR) sensor. (c) SEM image of the new composite element (CE) bit design, where each bit consists of high aspect ratio strips and maintains a consistent magnetic area. |
However, the original bit design6,7 was prone to forming multi-domainal states, which heavily limited encoding reliability and therefore also the total number of distinct bits. Here, we expand on the concept of coercivity tuning and present a new composite element (CE) bit design (Fig. 1c) that is capable of encoding up to 14 bits (16384 codes) with unique coercivity values to a high degree of accuracy. The CE bits consist of multiple strips with high aspect ratios and are engineered to enable a significantly wider coercivity distribution while maintaining sharp magnetisation reversal behaviour for reliable encoding. This makes the CE bit design highly relevant for the development of future microfluidics-based combinatorial chemistry applications, where each bit within a microcarrier can be individually encoded (when written from highest to lowest coercivity value) after each split-and-mix process. Compounds of interest could then be identified using multiplexed detection of the microcarriers' magnetic stray fields, all within a lab-on-a-chip platform. Here, we present the CE bit design and the intricacies that govern its encoding properties in order to demonstrate the reliability of the new barcode design.
Fig. 2 2D stray field measurements of original magnetic bit design,6,7 obtained by scanning an in-plane TMR sensor with its sensitive direction along the length of the Co elements over a 50 × 80 μm area. Scan taken at a height of 2 μm above the 3 bits with low aspect ratios. The images (top to bottom) show a microscope image of the microcarrier, the real component of the impedance sensor (X), its amplitude (R) and the phase (θ). |
It is evident that the domain breakdown observed for the low aspect ratio (low Hc) bits reduces the stray field signal amplitude for in-flow detection and risks information loss. Although high aspect ratio (high Hc) bits do not suffer from domain formation, their total magnetic volume is reduced, which limits the distance at which they can be detected in-flow.8 Moreover, a domain breakdown prevents sharp switching and broadens the unique coercivity value of each bit, further increasing the risk of unintentional switching of neighbouring bits when writing a desired magnetic bit code using magnetic field pulses.6
The new CE bit design is able to overcome all these limitations by creating elements consisting of strips with significantly larger aspect ratios coupled by dipolar interactions. Here shape anisotropy is able to dominate the magnetic behaviour within each bit, as illustrated by the difference in domain formation between a single element and a CE bit in Fig. 3. As coercivity scales inversely with strip width,21,22 such a design is also able to significantly widen the possible coercivity distribution, while the appropriate dipolar interactions and use of magnetically soft materials ensure sharp magnetisation reversal behaviour23 for reliable encoding. In the CE bit design the magnetic volume is maintained between bits by increasing the number of strips for CEs consisting of narrower strip widths, allowing for consistent magnetic read-out of each bit's stray field in-flow.
The in-plane easy axis coercivity (Hc) value of a patterned strips at set spacings is determined by22–24
(1) |
For a maximised coercivity range we therefore select a relatively thick film of 15 nm for both the Co and Ni80Fe20 CEs to illustrate the difference between materials with and without crystalline anisotropy, respectively. The pitch between elements should be sufficiently large (e.g. >5 μm) to prevent bits from interacting and to allow for in-flow stray field detection for set TMR sensors properties and rapid flow rates.8 By varying strip width, length and spacing we investigate their effects on the coercivity distribution and switching behaviour of the barcodes in order to optimise the CE bit design for reliable use in combinatorial chemistry based applications.
Patterns for the CEs were designed using CleWin 4 (WieWeb software v.o.f.) and exposed using a Leica VB6 UHR e-beam writer at a 100 kV beam voltage, and developed in a 5:15:1 ratio of methyl–isobutyl–ketone:isopropanol:methyl–ethyl–ketone (MIBK:IPA:MEK) for 5 seconds followed by a thorough IPA rinse and N2 gas dry. The Co (99.95%) and Ni80Fe20 (99.97%) sources were purchased from Goodfellow Ltd. and were both thermally deposited, at a rate of 0.025 nm s−1, using an Edwards Auto306 evaporator with a base pressure of 2 × 10−7 mBar to a thickness of 15 nm, each with a 5 nm Au (99.99%) capping layer to prevent oxidation. All growth heights were confirmed using atomic force microscopy (AFM). The hysteresis loops were measured by longitudinal magneto-optic Kerr effect30 (MOKE) on elements with appropriate pitch using a NanoMOKE magnetometer, produced by Durham Magneto Optics Ltd., capable of focusing the laser down to a 5 μm spot diameter. Each presented MOKE coercivity value was obtained by averaging 200 hysteresis loops measured on each of at least 16 identical barcodes.
The spacing between strips can itself further effect a CE's coercivity value and switching behaviour. Fig. 4 [bottom] shows Ni80Fe20 elements with strip widths between 100–800 nm, each varying by spacings of 50–400 nm. It becomes evident that decreasing the separation between strips decreases the overall element's coercivity (Hc) while maintaining consistently sharp switching behaviour, as seen in the inset hysteresis loops of elements with 400 nm strip widths. The magnetostatic coupling between strips is determined by the number of strips, their spacing and width, and will effect both the overall coercivity as well as the mode by which an element reverses. This can range from a sudden avalanche-like switch to a more gradual cascading of pairs of coupled antiparallel strips.26–28 In the later case, systems with stronger dipolar interactions, a closer spacing between strips can actually increase the element's coercivity,29 since the magnetisation reversal onset is due to domain formation at defects while the transition itself is a slow cascade-like switching of strongly interacting strips, resulting in a slanted hysteresis loop.
However, the varied strip spacings of the CE bit designs have sufficiently low coupling to give an earlier reversal onset at reduced separations due to a lower domain formation barrier. Domains typically occur at the strip ends and rapidly propagate through all neighbouring strips without forming antiparallel intermediate states during the reversal process. The fine tuning of the dipolar interactions at the higher coercivity elements becomes increasingly difficult, as a greater number of strips (to preserve the total magnetic volume between elements) tends to result in more gradual transitions. For our final CE bit design we select spacings equal to the strip width to ensure appropriate dipolar interaction strengths.
The benefits of using magnetically soft materials for coercivity tuning are further illustrated in Fig. 5, where stacked MOKE loops of Co and Ni80Fe20 elements reflect the coercivity range presented in Fig. 4. Crucial for reliable magnetic bit encoding are sharp transitions where magnetisation is dominated by shape anisotropy, reflected by sharp changes in Kerr signal. The Co elements produce rounded and overlapping loops that can send neighbouring elements into minor loop positions or even full transitions and therefore heavily limit writing reliability, as was noted for the original bit design.6,7 The Ni80Fe20 elements on the other hand, result in much sharper transitions that are well separated from one another. This is particularly evident in the bottom plot of Fig. 5, where the derivatives of the Ni80Fe20 CE Kerr signals show no overlap between bits. The broadening of the differential plots for high Hc elements comprised of narrower strips, corresponding to more slanted hysteresis loop, is due to the greater difficulty of balancing dipolar interactions for growing arrays of strips, due to the tendency of forming antiparallel states during the reversal process. With growing arrays of strips there is also an increased likelihood of dispersion in CE strip features, where edge effect and lithography defects become more relevant in the reversal process. Nonetheless, it is evident that it is essential to use magnetically soft materials for the CE bit design to ensure reliable encoding of large bit codes.
Fig. 6 can now be used as a reference for an encoding process, as the different amplitude changes in Kerr signal of the de-focused laser position can be used to precisely identify each switching bit of the three element system. Fig. 7 demonstrates such an encoding process, where a 101 bit code is written from a 000 initial configuration. The plots show all transitions that the three bit system undergoes for every applied field pulse, thereby locking in the desired bit states from highest to lowest coercivity value with decreasing field amplitude. The orange X indicates when the field sequence has been completed and the desired 101 bit code is written. The schematic of Fig. 7 illustrates the same process, where the number of intermediate transitions decreases with decreasing applied field amplitude. The encoding process is carried out at 1 Hz due to induction limitations of our electromagnet, whereas the bit encoding itself can be performed much quicker (i.e. MHz range) and is only limited by domain formation and motion.31 The de-focused MOKE technique can also be used to demonstrate encoding of longer bit codes, but visually distinguishing all individual Kerr amplitude changes becomes increasingly difficult.
Fig. 7 Demonstration of controlled encoding of a 101 bit code from a 000 initial configuration in three neighbouring Ni80Fe20 CE bits using de-focused longitudinal MOKE, where distinguishable amplitude changes in Kerr signal correspond to the magnetisation reversal of each bit (as illustrated in Fig. 6). [Left] Normalised variation of Kerr signal with respect to applied field over time (at 1 Hz), showing required sequence of switches to write the desired 101 code. [Right] Hysteresis loop of the same encoding process, with a corresponding animation of the three illuminated bits undergoing all switching steps for each applied field pulse. The orange X indicates when the field sequence is completed and the desired 101 bit code is written. |
The scaling advantages of bit encoding for library generation using combinatorial chemistry can be modelled by a Poisson distribution using the assumptions of consistently even (50:50) split-and-mix processes, a 6σ accuracy in each chemistry step and a 2σ accuracy in every encoding step. This gives a joint failure probability of P = 1.05 × 10−6 for which we can find the appropriate level of degeneracy (λ), indicating the average number of tags per end state that are required at the onset to guarantee that every split-and-mix compound and bit code is realised in the assay. Using the equation33P = 1 − (1 − e−λ)2n where λ = N/2n, we can determine the total number of tags (N) required for a set number of combinatorial chemistry and bit encoding steps (n). Table 1 illustrates the exponential scaling advantages in library generation with the linear scaling of split-and-mix steps. Therefore, using 384561 tags (with 14 bits) in a 14 step split-and-mix process will ensure that at least one or more tag carries every possible compound (16384 in total) with the correct bit code. Encoding in-flow is also possible since the time scales for Néel rotation34 are many orders of magnitude below that of Brownian motion.
Steps (n) | Num. compounds | Degeneracy (λ) | Num. tags (N) |
---|---|---|---|
1 | 2 | 14.5 | 29 |
5 | 32 | 17.2 | 552 |
10 | 1024 | 20.7 | 21196 |
14 | 16384 | 23.5 | 384561 |
Since a 3′′ Si wafer can hold several million microcarriers (100 × 20 μm footprint) the number of required microcarriers can easily be met at low cost. Mass fabrication of feature sizes down to 50 nm can already be accomplished by existing semiconductor lithography procedures, such as interference lithography. Based on material and running costs of the bi-functional microcarrier fabrication procedure19 we estimate a cost of $100 for 4 million tags on a 3′′ wafer, which can be further reduced when using industrial wafer sizes.
As the true advantage of magnetically encoded microcarriers is scalable library generation and not rapid diagnostics, compared to for instance nanoparticle-based applications, the relatively large size of the microcarriers is not a limiting factor. Since the area of the tags is comparable to the spot sizes used in printed microarrays, optically intensive techniques such as fluorescence detection can still be used to perform quantitative analysis. Multiplexing can then rapidly identify compounds of interest based on the magnetic stray fields of the microcarriers' barcodes in-flow.33 For an alternative to fluorescence detection, it has also been shown35 that it is possible to incorporate Au gratings on micron sized carriers to detect binding interactions using grating-coupled surface plasmon resonance (GCSPR).
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