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[[File:Adup_sense_logo.PNG|200px|thumb|right|Logo of AdUp SenseEPFSens]]
== EPFSens 2019 ==
== Method ==
Extraordinary optical transmission (EOT). An optical technique uses is used which utilises a chip functionalized with capture antibodies and gold nanoparticles covered with detectionantibodies. Both antibodies target Humira. When the drug is captured by a gold nanoparticle close to the array, thisit induces a local change in refractive index. In practice, the intensity of the transmitted light is reduced at these locations.This variation of light intensity can be easily is measured using a camera and used to quantify the drug concentration.
==Molecular Recognition ==
The EPFSens bioassay, is designed as a 1-step ELISA-like sandwich assay. It uses two monoclonal antibodies, eachrecognizing a different part of the target analyte. We decided to use monoclonal Monoclonal antibodies were used to ensure high specificityand little background because of due to their inability to bind more than one antigen through their epitope. We selected twoanti-adalamimuab (anti-ADA) antibodies from GenScript, the capture antibody (clone 15C7) is the one at the surface ofthe chip. The detection antibody (clone 3C2) is the one bound to the signal generating particle [1] [2].The detection antibody is attached onto gold nanoparticles (Au-NPs) coated with N-hydroxysuccinimide (NHS) [3].Those Au-NPs are commercially available (by Cytodiagnostics) and the attachment is performed following manufacturerinstructions.In order to conduct the assay, additional reagents are required and mixed in two specific buffers. First, a blocking buffercomposed of bovine serum albumin (BSA) diluted a hundred times in phosphate buffer saline (PBS). This buffer is usedto activate the capture antibodies located at the surface of the chip. Secondly, we prepare a reaction buffer composedof NaOH (50 mM), BSA (1%) and Tween 20 (2.5%) in PBS. The role of this reaction buffer is to ensure an appropriatebasic pH of approximately 9, to limit non-specific interactions, to maximize the binding and to generate a signal.Note that for the plasma samples, we are still optimizing the blocking buffer. We tried with PBS and 5% of nonfat drymilk (Sigma).
Two anti-adalamimuab (anti-ADA) antibodies were selected from GenScript, with the capture antibody (clone 15C7) being at the surface of the chip and the detection antibody (clone 3C2) being bounded to the signal generating particle.
The detection antibody is attached onto gold nanoparticles (Au-NPs) coated with N-hydroxysuccinimide (NHS).
The Au-NPs are commercially available (by Cytodiagnostics) and the attachment is performed following manufacturer instructions.
In order to conduct the assay, additional reagents are required and mixed in two specific buffers. First, a blocking buffer composed of bovine serum albumin (BSA) diluted a hundred times in phosphate buffer saline (PBS) is required. This buffer is used to activate the captured antibodies located at the surface of the chip. Secondly, a reaction buffer composed
of NaOH (50 mM), BSA (1%) and Tween 20 (2.5%) in PBS is to be prepared. The role of this reaction buffer is to ensure an appropriate basic pH of approximately 9, to limit non-specific interactions, to maximize the binding and to generate a signal.
== Physical Transduction ==
The detection principle used in this project is based on the variation of extraordinary optical transmission (EOT) intensitythat a gold nanohole array (Au-NHA) presents when a gold nanoparticle (Au-NP) of 100nm diameter is located in close
vicinity of a nanohole. Our Au-NHA chip is functionalized with anti-ADA antibodies (clone 15C7) on specific spots as in
Figure 1.a and illuminated with 660nm light to generate the EOT via a plasmonic effect at the gold surface. This effect
