EPFSens 2019
EPFSens is a team from École Polytechnique Fédérale de Lausanne competing in the SensUs 2019 event. For SensUs 2019, EPFSens investigated the possibilities for creating a biosensor which is able to measure the concentration of Adalimumab. The full TRD can be found [ via this link]
Method
Extraordinary optical transmission (EOT). An optical technique is used which utilises a chip functionalized with capture antibodies and gold nanoparticles covered with detection antibodies. Both antibodies target Humira. When the drug is captured by a gold nanoparticle close to the array, it 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 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, each recognizing a different part of the target analyte. Monoclonal antibodies were used to ensure high specificity and little background due to their inability to bind more than one antigen through their epitope.
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 is based on the variation of extraordinary optical transmission (EOT) intensity that a gold nanohole array (Au-NHA) presents when a gold nanoparticle (Au-NP) of 100nm diameter is located in close vicinity of a nanohole. The Au-NHA chip is functionalized with anti-ADA antibodies (clone 15C7) on specific spots and illuminated with 660nm light to generate the EOT via a plasmonic effect at the gold surface. This effect is enhanced by the nanohole array found on the chip. Without any sample on it, the chip has a uniform transmission. A sample solution containing the target analyte (adalimumab), activated Au-NPs functionalized with detection antibodies (clone 3C2) and reaction buffer is put on the chip. The adalimumab protein will eventually bind to the capture antibodies fixed on the Au-NHA surface. The bounded adalimumab alone induce only very little change in intensity of the extraordinary plasmonic transmission which cannot be detected with a standard camera. Instead, the binding of a Au-NP on top of the adalimumab triggers the detection. The vicinity of this NP to the NHA induces a local change in refractive index, which strongly affects the plasmonic resonance peak present on the surface of the Au-NHA. The intensity of transmitted light at the NP locations will be reduced, thus making it possible to digitally measure variation of intensity on a far-field image (black dots will appear, representing a NP).
Cartridge
In the assay, the capture antibodies are deposited with a spotting machine (Scienion sci-FLEXARRAYER S3) on the gold nanohole array (Au-NHA). The spots have a diameter of 100 μm and are spaced every 300 μm. The machine is programmed to spot both the capture antibody used for the assay and a mouse antibody that is used as a negative control for reference. On the chip, a silicon rubber is placed to form a well. The sample is added inside and then a round cover slip is used preventing the evaporation of the sample. The volume of the sample is around 20 μm. Furthermore, to facilitate the insertion of the chip in the device, a single use holder as been cut in PMMA.
Reader Instrument
The reader’s dimensions are estimated at 42x30x24 cm. It is composed mainly of two parts. The hexagonal tower contains the optical setup as well as a slot for inserting the chip and a z-axis translation mount accessible to the user for focus adjustment. As for the base support, it contains the switching power supply for the LED and the raspberry pi used for user-machine interaction.
A user interface for our touchscreen is implemented and the user is able to choose to refer back to previously recorded results or make a measurement. The measurement process is simplified by a provided tutorial displayed with instructions on the handling of the cartridge and the result acquisition. The user is also guided during the focus adjustment before starting the measurement in order to ensure the best image quality for the analysis. The implemented software is continuously printing the image on the screen, as the user is changing the z-axis translation mount for focus adjustment. As part of the development of our prototype, it is intended to implement an autofocus algorithm so as to further simplify the user interaction. The main job of the software consists in acquiring the images of the assay over a certain time period and then uses image analysis algorithms to detect capture antibody spots locations, where the detection takes place. The device prototype has the added possibility for the user to visually check the position of the spots and, if needed, to adjust the detected circle position, but this step would ultimately only take place in the background without any user input. After spot detection, the camera takes regular captures of the spots, showing the increased binding of adalimumab on the nanohole gold array over time, which is translated by the increasing number of black dots on the captured image. The software measures the variation of the image intensity in time and outputs the detected adalimumab concentration, based on a well established calibration curve. The result is saved and printed on the screen.