Fabrication Of Tunable Long Range Ordered2D Portein Nanoarrays And Their SERRS Detection

Nanometer scale protein arrays have gained considerable attention nowadays dueto an increasing demand on biominiaturization and bioassay. For example, uniformprotein patterns were used in the research of microfluidics for living cell detection,cell adhesion, chemotaxis, cell apoptosis and cell purification. Multiple proteinpatterns were used in the study of fabricating chips of biosensor, monitoringorientations of cell growth, observing interactions of proteins. The protein arrays havebeen a exciting platform for bioassays in proteomics, drug discovery, and diagnostics.Although great progresses have been made in the field of protein-array-based bioassay,the detection method is mainly depends on the FL spectroscopy. FL spectroscopy issensitive, convenient and can be used for quantitative analysis, but also suffers fromthe shortage of photobleaching, which is prone to happen when increasing thecomplexity of the detecting system, due to an amplified inference from eachcomponent. Some supplementary methods are still needed to increase the detectionperformance on complex biosystems.Surface-enhanced Raman scattering (SERS) is an emerging spectral methodshowing great potential applications in materials, physics, environment and chemistry.Recently, the SERS research also demonstrates powerful applications in the detectionof trace amounts of proteins. The excellent features of SERS, such as high sensitivity,high selectivity, convenient, and being not affected by the presence of water, make itvery suitable to investigate biomaterials, both in component analysis and inquantification. For protein detections, the most important model is the immunoassaywhich is based on antibody-antigen immunoreactions. Dye molecules were usuallyused as tags on antigens or antibodies by anticipating FL detections of the obtained immunocomplex. In SERS, these dye molecules can also be probes as Ramanscattering centers. Meanwhile, when the excitation lasers are selected properly to beequal to the electronic energy levels of dye molecules, additional enhancement up to10^-610^-8can be achieved due to a resonance effect. Thus, the optimal sensitivity ofSERRS can be on single molecule level.Herein, we aim at combining SERRS detection with nanometer scale proteinarrays and inspecting the performance of sensitivity and quantification on proteinsarrays. We employ both the lithography technique and the template method to prepareordered two-dimensional protein arrays, and with the help of SERS spectra, thedetection limit of labled protein can achieve as low as1ng/mL. Our study is outlinedas follows:1. Nanoscale protein arrays are prepared by using an etched PS template. Thisprotein arrays have round shape and can be directly used for immunoassay. Someglass slides submerged in aldehyde is initially prepared, modified with antibodies, IgG.Then PS arrays are self-assembled on these slides with the Langmuir-Blodgett method.The PS template pattern is transferred to the Human IgG substrate using an etchingprocess-slides are exposed to O₂plasma for90s. The PS nanoparticles are thenwashed away using phosphate buffered saline solution. Next, the slides are dippedinto bovine serum albumin solution to ensure that the anti IgG would bond only to theHuman IgG. At this moment, a patterned protein chip is obtained. When used forprotein detection, the protein chip could be immersed into labeled specificity antigensolution. Here we chose Fluorescein isothiocyanate anti-human IgG. After washing,only bonded antigens remain. Fluorescence microscopy and SERS is used tocharacterize the samples. The SERS spectra intensity shows correlation with theconcentration of anti-human IgG.2. Two kinds of protein molecules (antigens) were used as building blocks. As aresult, dual-component biocompatible surfaces with alternate immunoglobulinmicropatterns were fabricated. The employed antigens included human IgG and rabbitIgG, which composed nanometer scale surface arrays on the surfaces. The antigenswere identified specially by immunoreactions with labeled antibodies ofFITC-antihuman IgG and TRITC-antirabbit IgG. The immune responses wereconfirmed by confocal FL microscopy. A study on the sensitivity and quantificationwas done by using SERRS spectroscopy. The obtained SERRS spectra showed satisfactory resolution in the multi-component detection objects. No interference wasobserved from inner-or interactions of detecting molecules. The detection limits forboth of the antigens reached to as low as1ng/mL, which was comparable to FLmethod. Meanwhile, a good linear relationship between SERRS peak intensity and thelogarithm of antigens' concentrations (from1ng/mL to10μg/mL) were observed. Theresults demonstrated that SERRS is a very promising detection technique formulti-component immunoassay, and has great potential applications in biotechnologyand biochemistry.3. By means of SEF, ultra-low concentration of labled IgGs can be detected aslow as1pg/mL. The SEF structure is fabricated as follows. Patterned Ag film isconstructaed with the help of PS templates. Then antigens can be selected obsorbedon the surface of Ag nanoarrays. An immunoreaction is taken place on the as preparedantigen arrays. The patterned Ag film act as the enhance substrate, could provide thefluorescence enhancement of labled molecules. Meanwhile, nanoscale ordered patterncould provide an effective enrichment of proteins. While compare to ordinary Ag film,patterned nanoarrays can achieve more34%enhancement. The structure preparedhere has more intuitive results, and has great potential applications for proteindetections.4. Semiconductor nanoparticles, especially titanium oxide (TiO₂) with excellentoptical property, have been broadly used in industry field, such as environmentalcleanup, photocatalysts, solar cells, etc. Meanwhile, TiO₂material has extraordinarybiocompatibility using as implant materials. Nanoscale TiO₂also severed asbactericidal material in medicine. In this work, enhanced Raman spectra ofCytochrome c (Cyt c) absorbed on TiO₂nanoparticles are observed. According to theinteraction between Cyt c and TiO₂nanoparticles, Raman spectra are quite differentfrom it on Cyt c bulk. The position of the peaks shift and the relative intensity changeas well. For the quantitative analysis, the concentration of Cyt c is adjusted from10-4M to5×10^-7M. A concentration–dependent relationship with intensity is found.Meanwhile, the detection limit of Cyt c can achieve as low as5×10^-6M. It is possibleto predict semi-quantitative analysis the concentration of Cyt c.