Microfluidic devices and nano materials play a key role in the emerging biological applications such as lab-on-a-chip, which include pretreatment and purification of protein sample, and diagnosis and therapy of disease. Recently, microfluidic devices for biological sample pretreatment have become important in the design of analytical devices for high-throughput applications such as functional validation of proteins and peptides in large-scale proteome characterizations. In addition, nano materials for diagnosis and therapy are young and rapid emerging field such as point-of-care (POC) biosensor, tumor pretargeting agent, drug delivery vehicle and diagnostic imaging agent. In this study, micro-dialysis ($\mu$ -dialysis) and microfluidic free-flow isoelectric focusing ($\mu$ -FFIEF) systems were developed for the protein sample preparation, and porous AAO-integrated microfluidic system for enzyme-linked immunosorbent assay (AAO- $\mu$ -ELISA) and diacetylene-modified single-walled carbon nanotube (PDA-SWNT) were prepared for the nano materials for biological applications.
First, $\mu$ -dialysis system was developed for the removal of urea or guanidine hydrochloride (GdnHCl) from red fluorescence protein (RFP) extract. In this system, affinity streams facilitated by copper (II) ion was employed in the five lamina streams expressed by ‘affinity stream (AS)<->buffer stream (BS)<->sample stream (SS)<->buffer stream (BS)<->affinity stream (AS)’. In this system, were employed at outer position of the buffer streams (BS). Simple diffusion expressed by ‘BS<->SS<->BS’ showed about 55% of urea removal with 85% of protein recovery from SS containing protein. Diffused Cu(II) ions into the BS react with urea or GdnHCl molecules. Affinity diffusion system resulted in about 72% desalting efficiency while maintaining the recovery of protein similar to the simple diffusion device. Furthermore, fluorescence intensity and spectral quality was also improved.
Second, $\mu$ -FFIEF system was demonstrated for the biological sample concentration and separation using branched electrodes and glass coating with sol-gel method. Used protein sample was green fluorescence protein (GFP) and RFP, which have 5.8 and 6.7 in isoelectic point (pI), respectively. These proteins were successfully concentrated and separated within 30-120 sec under the pH gradient using 1 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer with 1.5 V DC/cm.
Third, AAO-$\mu$ -ELISA system was proposed for rapid and throughput diagnosis of hepatitis B virus (HBV). AAO was fabricated by anodizing aluminum deposited on the microscope glass slide to form hexagonal pore arrays of pores with 80-120 nm openings. The pores were coated with $SiO_2$ by sol-gel method, and functionalized by primary antibody for HBV. The assay using AAO- $\mu$ -ELISA was successfully carried out with comparable results with the conventional ELISA system within 15 minutes. From the results, rapid assay based on higher mass transfer rate and thermal conductivity was enabled by AAO nano-pore structure. In deed, AAO embedded in microfluidic device offers potential opportunities for the biochemical applications.
Finally, we studied the functionalization of single-walled carbon nanotubes (SWNTs) for biochip, drug delivery vehicle and cellular imaging agent using supramolecular self-assembly of PDA. SWNTs dispersed in water containing 10,12-pentacosadiynoic acid (PCDA) resulted in PCDA-wrapped SWNTs (PCDA-SWNTs). Consecutive UV irradiation converted PCDA-SWNTs into PDA-SWNTs with strong visible and near-infrared emission. For further chemical modification, biotinylation of PDA-SWNTs was carried out to give biotin-EDDA-PDA-SWNTs. The micro contact printing (MCP) of PDA-SWNTs and biotin-EDDA-PDA-SWNTs were demonstrated using covalent bond and biotin-avidin interaction, respectively. The biocompatibility test of PDA-SWNTs was performed by viability experiments using various cancer cells, which revealed that the PDA-SWNTs exhibited very low toxicity up to 31.3 mg/L in terms of pure SWNT concentration. Furthermore, PDA-SWNTs inside the cancer cells could be observed by near-infrared (NIR) microscopy.
In conclusion, we have developed effective lab-on-chip systems for proteomic sample preparation and diagnosis of HBV using u-dialysis and $mu$ -FFIEF systems, and AAO- $mu$ -ELISA, respectively. In addition, new nano materials with biocompatibility for biosensing, drug delivery and NIRF imaging were proposed by supramolecular self assembly of PDA on SWNT. The requirements for advanced microfluidic systems for biomedical applications can be simply formulated by having higher sensitivity and throughput. To increase these requirements, the incorporation of nano-structured materials, i.e., AAO and SWNT, into the microchannel as well as novel design and miniaturization of microfluidic device is a must. Increased sensing surface increase the sensitivity, shrink the devices. In conclusion, we have shown the possibility of an efficient solution to tackle some of the challenges in biomedical applications using microfluidic systems and nanometer-sized particles or structures in lab-on-a-chip platforms.
최근 부각되고 있는 프로테오믹 시료의 처리 및 질병의 진단과 치료와 같은 바이오 응용기술에 있어서 마이크로플루이딕 장치와 나노재료는 매우 중요한 역할을 하고 있다. 본 연구논문에서는 소프트리쏘그라피 및 poly dimethylsiloxane (PDMS) 리플리카 몰딩 기술을 이용하여 바이오 응용을 위한 마이크로플루이딕 장치를 제작하였다. 첫 번째로, 미세유체장치를 이용하여 적색형광단백질(RFP)로부터 유레아 (urea) 및 구아니딘 (GdnHCl)을 제거하는 미세투석 ($\mu$ -dialysis)을 수행하였다. 본 장치는 구리이온 ($Cu^{2+}$) 에 의해 촉진된 확산을 이용하여, 72% 이상의 유레아 및 구아니딘의 제거효율과 80% 이상의 단백질회수율을 보여주었다. 더불어, 단백질구조의 복원 및 정제 정도를 RFP의 형광특성과 질량분석 스펙트럼을 통해 확인 할 수 있었다. 두 번째로, 미세유체장치에서 단백질의 농축과 분리를 위한 자유흐름 등전집속 ($\mu$ -FFIEF)을 성공적으로 수행하였다. 각각 5.8과 6.7의 등전점 (pI)를 가진 녹색형광단백질과 적색형광단백질을 나뭇가지모양의 미세전극구조 및 채널구조를 가진 미세유체장치를 이용하여 30-120 초 이내에 분리 및 농축할 수 있었다. 최적의 pH기울기 형성을 위하여, 1 mM, 2-(N-morpholino)ethanesulfonic acid (MES)의 ampholyte 버퍼농도에서 인가된 직류전압은 1.5 VDC/cm이고, 이때의 전류밀도는 28.75 $mA/mm^2$ 을 유지하였다. 세 번째로, 고속 효소면역진단 (ELISA)법을 위하여 양극산화다공성알루미나 (AAO) 나노구조체를 포함하는 미세유체장치를 제작하였다. 목적질병인 B형 간염 (HBV)의 1차 항원의 수식을 위해 제작된 AAO는 80-100 nm의 공극을 가지며, 일차항원의 표면수식을 위하여 졸-젤 법을 이용하여 AAO표면을 $SiO_2$ 로 개질 하였다. 제작된 $\mu$ -ELISA AAO-미세유체장치는 검증실험에서 상용 ELISA와의 동일한 시험 결과를 보여주었고, 상용 ELISA에 비해 검사에 걸리는 시간을 최대 8배 단축시킬 수 있었다. 이러한 결과로 미루어, AAO나노구조체의 도입에 의한 향상된 물질전달 및 열전달 특성은 차세대 고속진단용 상용ELISA시스템의 구현에 적합할 것으로 기대된다. 마지막으로, 최근 기능화된 나노입자들 가운데, 독특한 광학적 및 물리적 특성과 통시에 생물학적 응용 가능성을 가진 탄소나노튜브 (CNT)의 관심이 집중되고 있다. 최근의 몇몇 연구자들에 의해 질병추적, 유전자 및 약물전달, 그리고 생체영상진단 등에 있어서 CNT의 응용가능성을 타진하고 있다. 본 연구에서는 초분자자기조립 (supramolecular self-assembly)을 이용하여 단일벽탄소나노튜브 (SWNTs)에 광학적 및 화학적 기능성 부여하는데 성공하였다. 이를 위하여, 10,12-pentacosadiynoic acid (PCDA)를 SWNTs에 결합하여 PCDA-SWNTs를 형성하고, 254 nm 파장의 자외선을 이용하여 PDA-SWNTs로 전환하였다. 합성된 PDA-SWNTs는 670-900 nm 영역의 근적외선 형광을 띄며, 물에 분산된 용액은 1년간 초기의 분산상태를 유지하였다. PDA 말단의 -COOH기는 $-NH_2$ 와 공유결합을 통해 아민이 패턴된 유리기판에 PDA-SWNTs의 미세패턴을 형성할 수 있었다. 또한, $H_2N-NH_2$ 브리지를 이용하여 바이오틴 (biotin)을 PDA-SWNTs의 표면에 수식함으로써, 아비딘 (avidin)유도체로 기능화된 미세패턴에 PDA-SWNTs를 특이적으로 결합시킬 수 있음을 보여주었다. 전처리를 하지 않은 CNTs에 비해 본 PDA-SWNTs의 생물학적 친화성은 다양한 종류의 암세포에 대해 매우 높은 친화성을 보여주었고, PDA 소포낭 (vesicle)와 달리 암세포에 고농도로 전달될 수 있는 특성을 지녔음을 공초점레이저스켄현미경 (CLSM) 및 근적외선형광현미경 (near-infrared fluorescence microscopy)를 통하여 확인할 수 있었다. 결론적으로, 본 논문에서는 생물학적 시료의 전처리, 분리 및 정제, 그리고 진단의 일련의 과정에 대한 예시와 가능성을 제시하였으며, PDA-SWNTs를 통하여 나노플루이딕스 및 나노디바이스에 대비한 나노물질의 새로운 가능성을 제시하였다.
