A Solid Substrate And Surface Strategies For Generating Protein Biochips For IVD Application

A biochip is a collection of miniaturized test sites (microarrays) arranged on a solid substrate that permits many tests to be performed at the same time in order to achieve higher throughput and speed. Typically, a biochip's surface area is no larger than a fingernail. Like a computer chip that can perform millions of mathematical operations in one second, a biochip can perform thousands of biological reactions, such as decoding genes, in a few seconds. Using technologies developed for the production of DNA microarrays, thousands of proteins can be spotted onto a chip. Subsequently, a biological sample can be spread on the chip, and binding proteins can be identified. The protein of interest can then be analyzed using techniques such as fluorescence imaging, time-of-flight mass spectrometry, and peptide mass fingerprinting. Such protein biochips promise a fast, high-throughput means to profile disease-related proteins or to study protein-protein and protein-drug interactions, which was previously only possibly using methods such as western blotting and enzyme-linked immunosorbent assays (ELISA).Although the advantages offered by such protein microarrays parallel DNA microarray technology, in particular with respect to the requirement of only tiny quantities of precious and expensive samples and reagents, the analogy with DNA arrays is more apparent than real. Constructing protein arrays requires more steps and is more complex than the generation of DNA microarrays. There are some particular difficulties of protein chip. Firstly, the sensitive nature of proteins, which often results in (partial) denaturation upon chemical treatment and immobilization; 2, unlike DNA, The protein cannot be biosynthesized in large scale.3, the limited specificity and affinity of antibodies to certain antigen.4, the difference abundance of different proteins in biological sample.5, cross-reactivity between antibodies and antigens.Elisa-based Protein chip of low density will be valuable in clinical diagnosis. There are already several commercial multiplex Elisa products. ELISA with high stability and sensitivity is a classical protein quantification tool usually performed on 96-well plate. The advancement of microscale robot spotting techniques and microarrayer allows for the miniaturization of the high-throughput ELISA.One of the paramount challenges of manufacturing a viable protein chip is the correct choice of a solid surface and the development of surface chemistry that is compatible with a diverse set of proteins while maintaining their integrity, native conformation, and biological function. For clinical use, the material should be low cost, easy to process, stable, commercial available, with high physical and chemical homogeneity. A desirable protein chip surface should meet the following requirements:1,binding enough protein or probe stably; 2, keep the biological function of the binding protein; 3, protein attachment on the chip should be controlled with respect to chemical selectivity;4, the background of the material itself should be low enough.。Poly (dimethylsiloxane) (PDMS) is the choice of material for a wide range of applications, because PDMS has many advantageous properties such as chemical inertness, no toxicity, ease of handling, and commercial availability. We believe surface modification of PDMS will be a cost-effective and time-saving strategy, if a facile method for surface modification can be developed, since surface modification retains the desired bulk properties of PDMS and reveals the need for new material development. A number of strategies have been developed for PDMS surface modification, which can be divided into two categories, namely physisorption and chemical coupling. Chemical coupling is stable but is difficult to achieve because PDMS is chemically inert, which is ironically one of its merits. Ma HW demonstrated a simple yet effective method to realize permanent and functional surface modification of PDMS. The herein method relies on the creation of iPDMS and subsequent SI-ATRP from iPDMS, which renders PDMS tunable surface properties, for example, from very hydrophilic to very hydrophobic. This combination of iPDMS and SI-ATRP makes possible the application-directed surface modification of PDMS. This cost-effective method will improve the advancement of bioMEMS, microfluidics, and chips for cellular studies, where surface properties of PDMS plays an important role. In this research, we tried to construct a protein chip analysis system based on this new type support material. This protein chip will detect more than 10 tumor markers by sandwich immunoassay. The whole research work included preparation of iPDMS and surface modification, strategy of immobilization of antibodies, preparation of mixture of antigen standard, preparation of mixture of labeled antibodies, establishment of process protocol. The analysis performance of the protein chip system will meet the requirement of clinical application.PartⅠ:Preparation of iPDMS sheets and Surface ModificationThe main objective of this part of experiment was to prepare iPDMS with desired thickness, hardness, and proper surface modification for making antibody array. To prepare iPDMS, the component C (vinly-terminated initiator), was first mixed well with PDMS (Sylgard 184) precursor (A) and curing agent (B) at a ratio of 10:1:0.1 (A:B:C), which could react with hydrosilane hydrogens in the presence of Pt catalyst. This mixture was then poured into a 6.6×7.7 cm2 mold and cured at 80℃for 2 hours, resulting in a transparent elastomer. The resulted iPDMS was then subjected to surface modification via surface initiated polymerization (SIP). The SIP reaction mixture had a mole ratio of OEGMA526/CuC12/Bipy/AscA=20/1/2/1, with a feed [CuC12] at 2.76 mM. SIP was conducted under argon protection and continued for 120 min at～25℃. The now poly(OEGMA) coated iPDMS sheets were further functionalized by incubating in an aqueous solution of 1 M bromoacetic acid and 2 M sodium hydroxide for overnight to generate terminal carboxyl groups, followed by washing with milli-Q water for three times, dried under flowing nitrogen and stored at 4℃. The main property of resulting iPDMS: Young's modulus E∽2.12 MPa and contact angleθ∽□□°±0.4°). The thickness of poly(OEMGA) was 311.4±4.8A. The above properties were similar to the reported.PartⅡ:Preparation of Protein microarray and parallel sandwich immunoassayThe protein chip system includes antibody-arrayed protein chip, mixture of antigen standards, mixture of HRP-labeled antibody, chemiluminescence substrates and other reagents. It also includes the reaction protocol and signal detection machine with data processing software.The polymer coated, COOH functionalized iPDMS was first immersed into an aqueous solution containing 0.1 M EDC and 0.1 M NHS for 30 min. A HD-2003A noncontact plotter was utilized to array capture antibodies (i.e., array solutions) on either NC or iPDMS membranes at 24℃and 45% humidity. The configurations of protein microarrays as well as the contents of array solutions, probing solutions and detection solutions were dependent on the experimental needs and they were presented along with data analysis.We constructed an iPDMS-based protein chip quantification system for 12 tumor markers including CA19-9, CA242, AFP, NSE, CEA, CA125, CA15-3, CA72-4, c-PSA,β-HCG, SCC and CK19. The chip reader was a cooled CCD camera and the software is Bioca 5.0. On iPDMS, there was no cross-reactivity happened between the paired antibodies of certain antigen and other antigens. The calibration curve of each tumor marker represented a reliable concentration measurement of each tumor marker. Due to the elastmeric nature of iPDMS, each well is tightly sealed upon clamping to sustain all the incubating and stringent washing operations. Compared to NC, iPDMS requires no blocking step and the washing frequency is reduced from 4 times to 1 time.Part III:Optimization of Process of preparation of the antibody microarray and ELISA processing protocol.There are many challenges of manufacturing a viable protein chip with high quality. At least, optimization of solid supporting materials and surface chemical, spotting buffer and process protocol will improve the analysis performance of the protein chip. Hydrophilicity of the surface is one of the most important factors that affect the quality. Surface with too high hydrophility will cause coffee-ring of the spot, and that with too high hydropopholicity will interfere the protein binding. The contact angle range of 42°-48°is suitable for IgG binding. The spotting concentration of anti body should lower than the saturated load amount, or the extra unbinded protein will be spit out and bind to the undesired area. The saturating load concentration of the immobilizing antibody of each tumor maker is around 0.2mg/ml. Some reagents such as 1% glycerol,0.1% triton-100 and 3% Mannitol in the spot buffer will improve the spot morphology, antibody stability. After antibody spotting, the protein chip should be protected by N2 at 24℃and 45% humidity for more than 24 hours. As to the process protocol,0.1% triton-100 was added to the chemiluminescence substrate to help the fluid to cover the iPDMS surface quickly and then keep resting. This improvements eliminated the smears around the spots.PartⅣ:Analytical performance evaluation of the optimized Protein chip quantification systemAs an IVD product, the most important is the quality of the kit. The protein chip quantification system should meet all the requirements for clinical application. The performance evaluation includes LOD, dynamic range, specificity, precision, stability and accuracy. The character of iPDMS of nonspecific protein adsorption to an "absolute" zero level enables the protein chip achieves a limit-of-detection (LOD) as low as 20 pg mL-1, which is sufficiently low for most current clinical diagnostic applications. The protein chi p exhibited coefficient of variation (CV) at<15% of both inter-assay and intra-assay of each tumor marker, which outperformed clinical diagnosis standard (i.e.,<15%). The stability test showed shelf-life for the microarray was six-month in minimal. The reactivity of microarray will be stable at least for one week under 37℃, which is necessary for transportation. To evaluate the accuracy of the protein chip, we tested some patient sera. The values of hospital were obtained by routine single indexed immunoassay and were used for reference purpose only. For 32 tested samples (from multiple iPDMS sheets), all were confirmed to be positive although the absolute values were different. We are conducting large scale experiments and will report them in the future.