Effective molecular recognition and highly specific chemical interactions between the receptor (sensor) and ligand (target molecule) determine the performance of biosensors;in other words, the usefulness of a biosensor is governed by the quality of the affinity reagent. The invention of hybridoma monoclonal antibody (mAb) technology opened the paradigm of affinity reagents, and since then, there have been many significant breakthroughs including bacteriophage display, cell-surface display, mRNA display and aptamers to name a few. However, the current technology to obtain affinity reagents for new molecular targets continue to be limited by the high cost, lengthy development time, limited availability, and often inadequate biochemical properties (e.g. affinity, specificity, stability, durability) for many applications. This rings especially true in advanced diagnostic and proteomic applications, where the demand for new reagents far exceeds the rate at which they can be produced. Aptamers are functional binding molecules composed of nucleic acids which are selected from combinatorial nucleic acid libraries by in vitro selection through a process known as Systematic Evolution of Ligands by EXponential enrichment (SELEX) 1, 2. Unlike traditional reagents (i.e. mAb), aptamers provide a significant advantages as biosensors because they are chemically synthesized, can be evolved for affinity as well as specificity, and they undergo reversible de-naturation, thus making them potentially ideal for field use. However, current methods of SELEX usually require intensive manual labor (8-15 rounds of selection) and time (multiple weeks). Technically, the challenge in isolating high affinity binders originates from multiple factors;first, due to the fact that the starting library contains significantly fewer high affinity binders compared to non- and low-affinity binders, they """"""""out-compete"""""""" the high-affinity binders due to mass-action, especially during the crucial, early rounds of selection. Secondly, the non specific binding of the aptamers to the molecular target, the solid support or the separation apparatus degrades the efficiency of molecular partitioning. Finally, using traditional methods of selection, such as affinity columns and nitrocellulose membranes, the partition efficiency in separating the high-affinity binders from non/low-binders is limited, necessitating many rounds of selection. Thus, in order to accelerate the affinity reagent generation, a fundamentally new technology platform to screen molecular libraries must be developed that can overcome these key challenges. Toward this end, our laboratory recently developed a novel system for high performance magnetophoretic separation called CMACS where we use micropatterned ferromagnetic materials in microfluidic channels to generate a highly controlled magnetophoretic force field that separates superparamagnetically labeled molecules from complex backgrounds with unprecedented efficiency. This device allows the use of very small number of magnetic beads (<106 per selection) and target proteins (down to ~ 20 attomoles) enabling exceptional stringency such that low-affinity binders, do not """"""""out-compete"""""""" the high-affinity binders due to mass-action. Furthermore, the partition efficiency (PE) of CMACS to be 1-2 orders of magnitude higher than those achieved by capillary electrophoresis (CE). These advantages, combined with our bead conjugation chemistry, which inhibits non-specific binding have enabled us to generate aptamers with low nanomolar affinities to protein targets, in a single round of selection within 30 minutes of separation. Building on these foundations, the main goal of this work is to: 1) optimize the M-SELEX process and verify that affinities of the aptamers can be further improved with more rounds of selection, and 2) test the generalizability of the M-SELEX system by generating aptamers for multiple protein targets. The success of this project will have significant impact in multiple areas of biotechnology because it will enable the generation of high performance affinity reagents at an unprecedented speed with minimal labor and cost.
Detection of target viruses with traditional surface marker-based immunoassays (i.e., ELISA) using monoclonal antibodies (mAb) suffer from major drawbacks because 1) a new mAb typically takes more than 6 months to generate, 2) many of the antibodies cross-react across species and subtypes, thereby compromising the detection specificity, and 3) it is extremely costly to develop arrays of antibodies that can provide a composite signature of the surface proteins. Aptamers are functional binding molecules composed of nucleic acids which are selected from combinatorial libraries by in vitro selection. Unlike mAbs, aptamers provide a significant advantages as biosensors because they are chemically synthesized, can be evolved for affinity as well as specificity, and they undergo reversible de-naturation, thus making them ideal for field use. Expanding on our previous work on ultrahigh performance Microfluidic-SELEX, this proposal seeks to develop the Microfluidic Viral SELEX (MVX) technology which will be capable of generating a set of high affinity aptamers which are specific to the target virus, so that a composite signature of viral surface proteins can be generated on demand - rapidly, efficiently, reproducibly, and in disposable microfluidic devices. We envision that, when fully developed, the proposed MVX technology will be applicable to almost all viruses. However, in this R21 project, in close collaboration with a leading virology lab at UCLA, we propose to use two closely related herpes simplex viruses (HSV) as a model system. HSVs have complex surface structures and they will serve as a challenging, yet realistic models. If successful, the proposed MVX technology has the potential to fundamentally revolutionize the way we generate affinity ligands, and perform viral diagnostics.
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| Xiao, Yi; Plakos, Kory J I; Lou, Xinhui et al. (2009) Fluorescence detection of single-nucleotide polymorphisms with a single, self-complementary, triple-stem DNA probe. Angew Chem Int Ed Engl 48:4354-8 |
| Kim, Unyoung; Qian, Jiangrong; Kenrick, Sophia A et al. (2008) Multitarget dielectrophoresis activated cell sorter. Anal Chem 80:8656-61 |
