Nano-Integrated Suspended Polymeric Microfluidics (SPMF) Platform for Ultra-Sensitive Bio-Molecular Recognition of Bovine Growth Hormones

The development of sensitive platforms for the detection of biomolecules recognition is an extremely important problem in clinical diagnostics. In microcantilever (MC) transducers, surface-stress is induced upon bimolecular interaction which is translated into MC deflection. This paper presents a cost-effective and ultra-sensitive MC-based biosensing platform. To address these goals, the need for costly high-resolution read-out system has been eliminated by reducing the cantilever compliance through developing a polymer-based cantilever. Furthermore a microfluidic system has been integrated with the MC in order to enhance sensitivity and response time and to reduce analytes consumption. Gold nanoparticles (AuNPs) are synthesized on the surface of suspended microfluidics as the selective layer for biomolecule immobilization. The biosensing results show significant improvement in the sensitivity of the proposed platform compared with available silicon MC biosensor. A detection limit of 2 ng/ml (100pM) is obtained for the detection of bovine growth hormones. The results validated successful application of suspended polymeric microfluidics (SPMF) as the next generation of biosensing platforms which could enable femtomolar (fM) biomolecular recognition detection.


Fabrication of SPMF
For the fabrication of SPMF biosensing platform, SU-8 photoresist patterning technique was used to prepare two molds which were used further to fabricate the two thin layers of the cantilever. A schematic of a cantilever is shown in Figure 1-a. As it can be seen in this figure, the platform contains 4 layers including two thick substrate layers that hold the cantilever layers and two thin layers that are including: (1) top cover layer to close the channel with the thickness of t 1 and (2) a microfluidic-structure layer containing the buried microchannel with the thickness of t 2 .
Thick substrate layers are flat PDMS layers that can be fabricated easily by molding PDMS on a flat surface. However, for the fabrication of thin layers two SU-8 molds for cover layer and microfluidic-structure layer were fabricated and explained in detail here.
Preparation of top thin layer is a one-step standard SU-8 process as shown in Figure S1-A. Five molds with different thicknesses (t 1 ) were fabricated and used to prepare cantilevers in order to find the optimized thickness of the cantilever cover layer of SPMF. SU-8 was spin coated with five different thicknesses of t 1 =15 µm, t 1 =25 µm, t 1 =40 µm, t 1 =60 µm, and t 1 =105 µm on a cleaned 4" wafer ( Figure S1-A, step2) and then soft-baked at 95°C for 3-20 min. The soft-backed SU-8 was exposed to UV light followed by a post-baking process for 1-10 min at 95 o C ( Figure   S1-A, step 3). Afterwards, the SU-8 was developed in SU-8 developer. Finally the mold is hardbaked at 200°C for 15 minutes (step 4). The mold was silanized in vapor phase to enable the removal of PDMS cover layer from the mold after curing.
The procedure for the fabrication of SU-8 mold of µF-structure layer is schematically show in Figure S1-B. It starts with spin coating of SU-8 2010 to a thickness of h=15µm on a cleaned 4" wafer ( Figure S1-B, step 2) and then soft-baking at 95°C for 5 min. The soft-backed SU-8 was exposed to UV light followed by a post-exposure baking (PEB) at 65°C for 5 min as depicted in step 3. The PEB should be performed at a low temperature of 65°C due to two main issues: (i) Even at low-temperature PEB, an image of the pattern would be visible in the SU-8 2010 coated layer. These images can be used as reference to align the second mask. (ii) low-temperature PEB 3 avoids the appearance of bubbles on the un-exposed parts of SU-8. As a result, low-temperature PEB does not deteriorate the smoothness of first SU-8 2010 surface which is going to be used for the second SU-8 2035 spin coating. The second SU-8 2035 layer was spin coated on the first layer to the thickness of t 2 =45 µm followed by soft baking of the wafer at 95°C for 7 min (step 4). Then, the wafer is exposed to UV light followed by PEB at 95°C for 5 min. It should be noted that before the second UV exposure, the mask should be aligned to the previous patterns by using the visible image of the previous layer's features. This is a very important step as any missalignment will cause that the buried microchannel will not be at the center of the cantilever. In the next step, the mold is developed in SU-8 developer and the molds are obtained as shown in step 6 of Figure S1-B. Finally the mold is hard-baked at 200°C for 15 minutes. Similarly, the mold was silanized in vapor phase to enable the removal of PDMS from the mold after curing. After fabrication of SU-8 molds, the PDM was molded for the fabrication of PSMF. PDMS compound was prepared by mixing the base polymer (pre-polymer) and the curing agent (crosslinking agent) with the weight ratio of 10:1 followed by the removal of air bubbles in a vacuum desiccator. Substrate layers are fabricated with the thickness of 3-4 mm by using a simple flat surface as a mold. The layers were then peeled off from the surface and were cut into rectangular shapes. Thin layers of the SPMF were fabricated by using the PDMS thin layer fabrication method. The fabrication process for two thin layers of cover layer and µF-structure layer are shown schematically in Figure S1-C (a and b), respectively.

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The fabrication of cover layer and µF-structure layers are similar. First, PDMS was poured into the mold and then a semi-silanized glass slide was placed on PDMS to let the excess of PDMS to be squeezed out of the mold ( Figure S1-C, step 1). While keeping the pressure one the glass slide, the PDMS thin layer was cured at 80 o C for two hours (step 2). Afterwards the semisilanized glass slide along with PDMS thin layer can be easily peeled off from the mold as the mold was already fully silanized (step 3). Figure S1-C (c) shows the bonding of two thin layers to form a closed microchannel at the cross-section of cantilever. Figure S1-D shows fabricated thin layers on a semi-salinized glass slide.
After the fabrication of all the four layers, the top substrate was bonded to the cover layer by using oxygen plasma. Then the bonded top substrate carrying cover layer can be removed from the semi-silanized glass. Then the µF-structure layer was bonded to the cover layer and then the whole cantilever was removed from the other semi-silanized glass. Finally, the bottom substrate was bonded (using oxygen plasma) to have the whole PDMS cantilever platform. One important issue in the fabrication of the cantilever is the correct alignment of the cover layer and µFstructure layer before PDMS-PDMS bonding. Any miss-alignment in the bonding process will result in the leakage of the microfluidic system. All the processes should be performed under an optical microscope. Image of a fabricated SPMF is shown in Figure 1-c with inlet and outlet connectors.  The following in-situ reduction reaction has been suggested between the gold ions and crosslinking agent of PDMS 2 : Upon the introduction of the gold solution, the curing (cross-linking) agent of the PDMS will act as a reductant and initiate the gold nanoparticle to crystalize and grow into the PDMS polymer network as illustrated in Figure 3-a. The in-situ reduction reaction ensures high stability of the 7 AuNPs on the PDMS surface as the particles are embedded into the polymer and interact strongly with the polymer network and thus forming nanocomposite in the SPMF channel.
Indeed, our experiments verify that the leaching of particles can be ignored. However, the in-situ process is a slow process occurring over 48hrs because of the heterogeneous nature of the reaction. The amount of the cross-linking agent (curing agent) available for the reduction of Au 3+ is not enough and therefore migration of the cross-linking agent toward the PDMS surface during the reaction is another issue that explains the slow reaction. Possible migration of cross-linking agents of PDMS is schematically shown by dashed arrow in Figure 3-a.
After in-situ synthesis, heat treatment was used in order to improve the distribution of AuNPs in PDMS 1,2 . SEM images after the incubation process show that there are many aggregated AuNPs all over the surface. Annealing process has been optimized to reduce the particles aggregation to obtain more uniform distribution of particles on PDMS surface. For annealing process, the fabricated SPMF platform was kept at 300 ο C for 10 min. SEM images of the AuNPs inside the buried microchannel of the annealed sample showing uniform particle size with average size of 125±5nm as shown in Figure 3-b. The size distribution histograms were extracted from the SEM images using a data visualization modular program. The size distribution histogram of the nanocomposite shows a very narrow size distribution of the particle with less than 8% size variation (125nm±5nm). High uniformity of the synthesized particles makes the composite cantilever platform a suitable candidate for sensitive biosensing experiments.

Biosensing Protocol
Immobilization of Ab on the gold nanoparticles is carried out though functionalizing the nanoparticles with 5mM 11-mercaptoundecanoic acid (NanoThink® ACID11) in ethanol The antigen solution was introduced into the channel by using a syringe pump at a flow rate of 10µl/min. When the Ag solution is injected into the channel, they interact with the immobilized Ab molecules and induce a surface-stress which bends the cantilever. The biosensing protocol has been schematically shown in Figure S2. In this figure, the presence of synthesized AuNPs on the surface of PDMS in the microchannel is shown and through the biosensing protocol, Ab and then Ag have been immobilized on the surface of AuNPs.
After each experiment, the buried microchannel was washed several times with the 0.01% Tween solution and DI water and dried in an oven at 50°C to make sure that there is no more antigen/antibody immobilized on the AuNPs. After the washing process, the cantilever was used for another biosensing experiments.