Biosensors have been tremendous attentions in a wide variety of applications from fundamental cell/protein biology to industrial food processing. Recently, the significant efforts have been made using pat-terned biochip of functional proteins such as antibodies, enzymes, and DNA for high throughput screening tool. To enhance the immobilization and reduce the undesired adsorption, the self assembled monolayer (SAM) is widely used as biochip surface because SAM offers desired surface wettability (hydrophob-ic/hydrophilic) as well as proper functional groups to immobilize proteins. The SAM template-based fabricat-ing methods, which use pre patterned SAMs, have been explored to pattern the functional proteins and served as microarray platforms for biological research. Among the current methods of biological array patterning, such as micro-contact printing, particle lithography, and photolithography, the soft lithographic method using an elastomeric stamp is one of the most versatile approaches and can satisfy the requirements of high throughput and low cost. However, this technique has major problems, such as stamp deformation and lateral diffusion of the solution.
An essential requirement is reducing non-specific binding (NSB), protein adsorption outside the pattern, when proteins are immobilized on a patterned template for high sensitive detection and reducing detecting errors. Protein-repellent surfaces have been proposed to prevent NSB, including a SAM with poly-ethylene glycol (PEG) and a hydroxyl (-OH) as a hydrophilic functional group on the Si or gold substrate. Among the bio-fouling layers, interest in PEG SAM to minimize the interaction of proteins with the substrate and thereby suppress bio fouling due to the highly hydrated PEG chains has increased.
In the specific binding region, a hydrophilic NH2-terminated or COOH-terminated SAM has been widely used with various cross-linking steps for covalent bonding with proteins on a planar substrate. But the use of a planar substrate limits the number of biomolecules that can be attached on the specific region. A non-planar micro/nanostructure or silica micro/nanoparticle can be applied on the specific region to enhance the biomolecular-binding capacity and the detection sensitivity.
However, there are limitations in the application of previously developed compositions of specific and nonspecific layers for grafting proteins. Owing to the hydrophilic characteristics of both specific and nonspe-cific regions, a protein solution is generally immobilized by one of the following methods: the protein solution is dropped over the entire substrate, the protein solution is dropped and covered with a coverslip, or the sub-strate is dipped in protein solution during the protein reaction time. With each of these methods, protein con-tact with the background layer is unavoidable, and background NSA occurs, even when PEG SAM is applied as the background layer. Additionally, these processes are passive approaches to reduce NSB, and are ineffi-cient in terms of sample usage. The ideal solution for preventing NSB would be a substrate that causes selec-tive dewetting of protein solutions in specific regions; such a substrate would also provide the advantage of minimum sample requirement. Recently, due to their favorable optical, electronic, and chemical properties, gold nanoparticles (AuNPs) have been applied to enhance the binding of chemicals and the sensitivity of opti-cal measurements. Through their affinity with functional groups, such as SH, CN, or NH2, presented on the SAM surface, AuNPs are well suited to various biosensor applications. Development of AuNPs-enhanced bio-applications has been primarily focused on optimizing chemical-binding affinity or the optical properties for measurement.
In this paper, we propose the new microarray fabrication method based surface treatment, patterning process and protein grafting process.
In terms of surface treatment, we propose the new method of SAM coating process and layer composition of microarray chip. The previous SAM coating methods were liquid-phase process and vapor phase-phase process. The typical SAMs are organosilane reacting with oxidized surfaces and alkanethiol reacting with gold. The formation of organosilane SAMs were carried out by using liquid-phase procedure in hydrocarbon solvents such as toluene, hexadecane, etc. The major problem of liquid-phase procedure arises from the ability of the precursor to copolymerize in the presence of water, so obtaining clumpy and disordered layers on substrate surfaces. To overcome the problems, deposition from vapor-phase has been proposed because the lower molecular density in the vapor phase should decrease the extent of self-polymerization of the silane precursor. In case of alkanethiol SAM, both liquid and vapor phase deposition have been applied on metals and semiconductor surfaces, such as Au, Ag, Cu, Ni, GaAs, etc. However, Au is the preferred substrate for solution preparation, since oxide-free, clean, flat surfaces can be easily obtained in ambient conditions. Therefore, liquid-phase process of SAM formation must be considered for SAM-assisted applications.
Therefore, in this paper, we proposed the ultrasonic-assisted liquid-phase process for effective SAM coating. The prepared substrate was immersed in SAM solution in the vacuum desiccator by applying ultra-sonic wave at 200 watt power for 30 min to decrease the self-polymerization of the precursor. The ultrasonic wave was applied during the whole dipping time. Finally, the surface morphology and surface roughness were improved over 30% than liquid-phase process. The SAM coated substrates that were used in this paper were made by applying this ultrasonic-assisted process.
In the microarray chip, we propose the new background layer which is composed of mixed SAM con-sisting of a methyl-terminated (OTS) and methoxy-(PEG)-terminated SAM layer as the non-specific binding layer. The OTS SAM was used to apply the hydrophobicity for allowing selective dewetting of the protein solution at specific versus nonspecific regions. And the methoxy-(PEG)-terminated SAM was applied for re-ducing the NSB in the background region. The wettability and protein adsorption were analyzed according to the mixing ratios of OTS/PEG SAMs. The selective dewetting condition was evaluated using the contact angle difference between specific and non-specific regions. The protein usage and adsorption were compared with the standard method (dropping the solution and covered with coverslip over the entire substrate).
Finally, protein microarray was fabricated on 1:1 mixed SAM as the background layer by using selec-tively dewetting method. The NSB was reduced by 78% compared to even PEG layer without an additional blocking process. And the peak intensity was improved by 20% compared with the PEG layer. The streptavi-din and biotin interaction and antibody were tested for protein sampling.
Additionally, the non-planar pattern was applied using cysteine-tagged protein G-modified AuNPs that enlarged surface area and directly immobilized the antibody with proper orientation in the specific region. The change in surface topology also led to a change in peak intensity; the peak intensity on that nanoparticle-modified surface was increased by about 5 times compared to that on the planar surface. This experimental increase was much higher than the theoretical increase of the surface area. The reason could be that, when performing surface modification on nanoparticles in solution, the chemical reaction efficiency of the binding proteins is higher than that conducted on the planar substrate. As the dimensions of the antibody are in the nanometer range, a surface with nano-topological features similar to or smaller than the size of the protein may be sensed by the protein and affect its behavior and binding affinity
In the pattering process, the process considered with two ways: mass production and customized pro-duction. The mass production method was occurred by low temperature oxygen plasma with hard mask. For the SAM patterning, the SAM treated glass substrate was loaded in the plasma chamber with an invar pattern mask for local conversion of the terminal group. Given that the ionized atoms to modify the SAM layer and the radicals for surface activation in the plasma state have no directivity, the gap between the substrate and the pattern mask should be minimized to avoid pattern broadening. Consequently, an invar mask and a neodymium magnet were used to generate the magnetic force, and a glycerol layer was coated between the mask and the glass substrate to reduce the gap. The local conversion of the terminal functional group to the hydroxyl radicals occurs for cross-linking with organosilane SAMs. The used hard mask is not deformed, and thus the pattern mask is reusable. In addition, this allows the fabrication of many SAM templates at the same time in a plasma chamber, thereby satisfying the high throughput and low cost criteria.
In aspect of customized production, non-direct laser-induced plasma generation method was proposed for SAM array patterning. The chrome (Cr) 100nm coated glass by e-beam evaporation is set upwards on base plate with vacuum suction. The SAM coated glass is set on Cr coated substrate with a few micro gap with coated layer downwards. Laser beam can be focused on Cr coated layer without affecting on SAM coated glass as the upper glass coated with SAM is transparent. Focused Laser beam exposured on metal layer can generated plasma by ablation of metal surface. The pattering size could be controlled with changing the laer power and gap between the Cr coated substrate and SAM coated substrate. This method has merits as follows : fast process, low price laser source and maintenance, flexible spot size control. Finally, the uniform spot array from 10μm to 500μm was fabricated by applying proposed methods.
For the protein grafting, the semi-contact wiping process was newly applied for fast and simultaneous protein immobilization, and efficiently reducing the NSB and protein usage.
This proposed new methods are novel technologies for the actual application of microarray biochip fabrication. And we believe that this method will contribute to advances in low-cost, high sensitivity, and high-throughput biosensor array applications.
단백질 바이오 칩을 제작하는 데 있어 기존의 레이어 구성과 패터닝 방법, 단백질 grafting 방법의 한계점을 극복하고 바이오 칩의 상용화를 위해 본 연구에서는 새로운 개념의 칩 구성 및 공정을 제안하고 해결하였다
소수성/친수성 자기조립 단분자막을 혼합시켜 백그라운드 레이어에 적용하여 단백질 용액이 선택적으로 원하는 specific region에만 wetting되는 방법을 제안하였다. 기존의 수동적이며 재료 소모가 많은 레이어 구성에서 능동적인 선택적 wetting 방법을 적용하게 되면 백그라운드 레이어의 노이즈 감소 및 스팟 감도의 증가 재료 소모의 감소 등의 효과를 얻을 수 있다. 또한 스팟 부분에 골드나노 파티클을 사용함으로써 측정 감도를 더욱더 증가 시켜 칩의 재현성을 확보하였다.
기존의 느리고 고가인 공정에서 탈피하여 양산성에 맞는 마스크 저온 플라즈마 방식을 이용하여 패터닝을 수행하여 빠르고 값싸면서 한번에 많은 양을 제작할 수 있는 패터닝 공정을 제안하고 어레이의 크기 및 위치를 자유롭게 바꿀 수 있는 레이저 기반의 패터닝 방식을 제안하여 어레이 패터닝을 수행하였다.
단백질 용액을 grafting할 때 재료의 소모가 적으면서 빠른 wiping 공정을 제안하였다. 위의 방법들을 이용하여 다양한 단백질에 대해 실험을 수행하고 대면적 어레이 칩을 제작하였다.
이상으로 단백질 어레이 칩의 레이어 및 제조 공정에 대한 연구를 수행하였다. 앞으로 본 기술 실제 산업에 적용하여 항체 항원 반응 등을 실험하고 다양한 재료를 한 칩에 제작하고 테스트 하여 실제 산업에 적용할 계획이다.