Silicon Microarray Pin with Selective Hydrophobic Coating

We have recently demonstrated the viability of silicon microarray pins, taking advantage of lithographic and batch processing of silicon micromachining in comparison with conventional and serial processing currently used for commercial pins. The reduced spot size (< 50% in diameter, i.e., > 4X in array density), however, made such undesirable needs as preprinting more pronounced. Preprinting, a common practice in microarray printing, drains out the excess liquid formed outside the liquid channel during dipping. In this paper, we describe how surface wettability can be controlled and report its dramatic effect on the printing performance, for the first time. By making the exterior surfaces hydrophobic and the interior surface hydrophilic, the excess liquid outside the liquid channel is eliminated, and the advantages of siliconmicromachined pins are fully exercised. INTRODUCTION DNA contact printing microarray is technology to robotically print DNA/RNA clones onto a glass slide and subsequently hybridized to two differentially fluorescently labeled probes. This technology is popular due to the high expense of obtaining valuable clones. Hence it is preferable to produce arrays with wide representations on a single chip, so complete exploration the broadest possible set of genes or genome are possible; done both systematically and comprehensive. DNA robotic contact printing was originally developed by Dari Shalon and Patrick Brown at Stanford University[1]. It encompasses a contact pin that can deposit small quantities of genetic materials on a solid support. Such contact pins, now available commercially, are usually made out of stain-less steel pins which are expensive due to serial manufacturing step and end tips which are prone to bending if excessive force are applied over extended usage [2]. Recently, a wide variety of contact printing devices, fabricated differently from traditional stain-less steel rods, have arisen and shown to produce DNA microarray with high density. Innovative devices such as ceramic capillary tips from George et. al, stain-less fountain pen from Reese et. al, and electrowetting/de-electrophoresis driven silicon-based microcantilevers from Belaubre et.al. have all shown great promises to provide high density and high throughput array [24]. However, the aforementioned devices suffer from either durability, requirement to costume tooling of accessory parts, sensitivity to leveling of tip holder to slide platter, difficulty in drying due to the enclosed liquid reservoir, limited fluidic storage spaces or tip clogging. Moreover, solution waste due to excess liquid pickup is a major problem for all the devices today as the valuable liquids are undesirably attached to the outer surfaces of pins and create the need for print printing, 1 Copyright © 2004 by ASME We have previously reported design and fabrication of silicon micromachined pin, with length of 44.45 mm and width 1.40 mm, it can be conveniently placed into conventional pin holder with no additional parts necessary [5]. It has the versatility to pick up volume ranges from microliter to nanoliters and also possess ability to print printing droplet size as small as sub-micrometer to hundredths of microns. However, we have noticed during previous print runs that, similar to conventional contact pins, silicon micromachined pin also suffered from precious solution waste when the pins are transferred to the 384 well-plate for fluidic (RNA/DNA) solution pickup prior to printing. Aqueous solution has the tendency to cling onto (wets) the hydrophilic surfaces (silicon dioxide) of silicon pin due to surface tension, and remain in place during printing. Due to the presence of excess solution at the outer surface, silicon pin, like the conventional pins, prints a number of slightly larger spot size (2X) in the beginning of the run. The phenomenon persists until the solution is either drained out during initial print run (usually 10 to 20 spots afterward) or evaporated to the environment. Generally, it is common practice to do a pre-printing run to remove the excess solution to keep the spot size more consistent. In this paper, we report a method to selectively control the surface energy of the pin. By coating the outer surface of pin with hydrophobic material and leaving the inner surface hydrophilic, we aimed to minimize the need for pre-printing. We test two different hydrophobic materials, HMDS (hexamethyldisilazane) and OTS (octadecyl trichlorosilane), selectively coated on the exterior surface of silicon pin. FABRICATION The fabrication of uncoated silicon pin is reported else where[5]. Fig. 1. outline the general processing steps for selective hydrophobic coating of silicon micromachined pin. First, the silicon pins, after fabrication, were thoroughly cleaned in standard sulfuric acid / hydrogen peroxide wash (5:1) by chemically oxidize the silicon surface to silicon-oxide and render the entire pin hydrophilic to water. Next, our goal is to selectively coat silicon pin with hydrophobic and hydrophilic layers. The pin tip, as well as the liquid channel and reservoir (designated as “interior”) should remain hydrophilic to water, thus allowing capillary force to dominate fluidic pickup and delivery. The part that should be hydrophobic to water is the part that tends to attract unnecessary and excess water pickup during liquid loading, resulting in solution waste. It includes the top, bottom, and the side surface of silicon pin (designated as “exterior” area). Therefore, we devise a plan to successfully shield the inner area with photoresist by placing a 1μl drop of photoresist inside the reservoir. Capillary forces will carry the photoresist down through the length of the channel until it reaches the tip of the pin. Once its done, the photoresist are left dry in the oven for around 5 minutes in 100 degree C and the interior area are protected. Next, we tried to coat the exterior area of the pin with two different hydrophobic materials, HMDS (hexamethyldisilazane) and OTS (octadecyl trichlorosilane). The contact angles of water on vapor-deposited HMDS and immersion-coated OTS have been reported to be 80 and 112 degree, respectively. HMDS is a commonly used vapor coating technique to improve the adhesion between silicon wafer and photoresist. It is also hydrophobic in nature and easy to apply onto the silicon surface. OTS is the hydrophobic selfassembly monolayer coating technique commonly produced by immersion of silicon oxide surface into OTS solution. Since photoresist had successfully protected the interior surface from exposure to chemicals, only the exterior surface is exposed to the OTS and thus hydrophobic coating is selectively coated on the exterior surface of silicon pin. Finally, after OTS coating, the pins are placed in acetone/methanol/water bath to remove the photoresist and deprotect the interior surface. The interior surfaces, including the liquid channel and the printing tip, are left hydrophilic (SiO2). At this point, the pins are ready for use. PERFORMANCE The performances of hydrophobically-coated silicon pins are evaluated and compared to an uncoated silicon pin and a commercial stainless pin (ArrayIt CMP4) by the following criteria. Liquid loading: Fig.2 shows the effect of selective hydrophobic coating on silicon pin versus an uncoated pin in the liquid loading process. The coated pin and uncoated pin are both dipped into a 4 μl hemispherical 3XSSC droplet for 10 seconds and are withdrawn from the droplet for evaluation by video imaging. The uncoated pin, is left with solution and needs to be drained out before printing. This is especially pronounced at the channel opening surface. On the other hand, t he coated pin, due to its hydrophobic nature (HMDS) is free from liquid accumulation at its outer surface. This characteristic result in consistent liquid volume loading regardless of the sample volume left in the microplate wells and allows for significant saving with solution consumption. Preprinting: Since there is no sample adsorbed onto the outside surface of the pin, there is very little irregularity during the initial printing steps. In comparison, the initial irregularity is unavoidable when loading large volume of sample (>6 μL) in the current practice and calls for preprinting run until consistent 1. Start from bare silicon pin Side view Top view 2. Protect liquid channel with photoresist 3. Hydrophobic Coating (HMDS or OTS) 4. Deprotect liquid channel by removing photoresist Fig. 1. Process flow for general selective hydrophobic coating. 2 Copyright © 2004 by ASME spots are obtained[6]. Fig. 3 shows the diagram comparing the initial spots size with various contact pins. In this experiment, sample volumes of 10 μl oligonucleotide with 3XSSC buffer are used for loading. Uncoated silicon pin, due to its hydrophobic nature and geometry, tends to have more accumulation of excess solution in the beginning of the run. It requires approximately 30 spots run to drain out excess liquid before consistent spots are reached. On the other hand, the both hydrophobic coated silicon pins (HMDS and OTS) require little or no preprint since the 1 spot printed is already small. Consistent spot of approximately 50 μm nominal diameter are achieved. Spot diameter data is taken by evaluating the fluorescent image of printed spots. .Fig. 4. illustrates the image of preprinted spots. CONCLUSION Thanks to the superior control of liquid loading volume, the spot size is further reduced and the number of spots per run is significantly increased. Spot size ranges between 40-60 μm, depending on the size of the pin tip. With such a pin, total of 500 spots or more are possible per loading. 1 2 3 4 (a) Uncoated silicon pin (b) Hydrophobic coated silicon pin 1 2 3 4 Fig. 2. Liquid loading. Selective hydrophobic coating of Si pin results in better liquid loading efficiency. (a) Si pin, without any coating, accumulates additional liquids at its outer surface. It is especially apparent at the opening of liquid channel. (b) When coated with OTS, Si pin shows no excess liquid pickup at the