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General

Q: What reagents are supplied by Nanomedical Diagnostics?

A: The necessary amount of Nanomedical Diagnostics Quench 1 and Quench 2 are included to run measurements on all biosensor chips that are purchased from the company, including the 5 Biosensor Chips included with the Agile R100 system.

Q: What reagents do I need to supply to run measurements with Agile R100?

A: Biotechnology grade 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.0, 50 mM.

N-Hydroxysulfosuccinimide (sulfo-NHS; CAS No. 106627‑54‑7).

Ultra High Purity (UHP) grade N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; CAS No. 25952-53-8).

Culture or molecular grade 1X phosphate buffered saline (PBS) pH 7.4.

Culture or molecular grade deionized water (CAS No. 7732-18-5).

Q: How do I learn to use Agile R100?

A: One day of onsite training with a Nanomedical Diagnostics Field Applications Scientist who can provide training to an unlimited number of attendees on the usage of Agile R100 is highly recommended. The system also comes with an extensive User Manual for reference.

Q: What kind of experimental design or assay optimization help is offered by Nanomedical Diagnostics?

A: Field Application Scientists are on-hand to provide technical support or travel to the site to help with chemistry optimization or other questions related to Agile R100. Contact technical support to inquire!

Experimental

Q: How do I immobilize my target to the Agile R100 Biosensor Chip?

A: Current Agile R100 Biosensor Chips come with exposed carboxyl groups to covalently link target directly to the graphene surface. The most common linkage is to use carbodiimide crosslinker chemistry (i.e. EDC/sulfo-NHS). EDC and sNHS activate the carboxyl groups on the surface of the graphene to react with any free amine group in your target and form a covalent amide bond. Alternatively, His-tag proteins have been immobilized to Agile R100 Biosensor Chips, and immobilizing the target using nickel chemistry has been performed on Agile R100 Biosensor Chips. Additionally, you can start by using EDC/sulfo-NHS chemistry with a biheterofunctional crosslinker where one functional group is an amine and the other is the functional group favored for your target of interest. This makes Agile R100 flexible and adaptable to established research and development pipelines. Other binding chemistries are possible, but Agile R100 Biosensor Chips have not been thoroughly tested for other chemistries at this time.

Q: How many Agile R100 Biosensor Chips will I need for my experiment?

A: A single experiment (such as for a dose response curve of a biological interaction) will require 1 biosensor chip or more, and the number of biosensor chips is dependent on the biological interaction of interest. If the interaction between the immobilized target and analyte in solution fully dissociates (with or without regeneration techniques), up to 10 interactions can be measured using a single Agile R100 Biosensor Chip. If the interaction does not fully dissociate, 1 biosensor chip can measure a single interaction, and the number of biosensor chips needed will equal the number of interactions tested.

Q: How many times can I use an Agile R100 Biosensor Chip?

A: The Agile biosensor chip can be used for up to 10 measurements, depending on the stability of the immobilized target, stability of the analyte during the experiment, and success of analyte dissociation from the immobilized target.

Q: When I choose transistors in the Agile R100 software, is the data still being recorded?

A: Yes, when you choose transistors in the Agile R100 software, the data from every transistor continues to record. Only the data for the selected transistors is displayed, but all data is recorded.

Analysis

Q: Can I export my data to another software after performing a measurement?

A: The experimental data can be exported as a .csv file for further analysis using a preferred scientific graphing and statistics software (such as GraphPad Prism). All images (such as the Experiment Graph, Analysis Data, and Analysis Graphs) can be exported as .jpg files. Additionally, an Experiment Report PDF file can be generated that exports all the data from an experiment file, including the Protocol, Notes, Analysis Settings, Analysis Data, and Analysis Graphs.

Q: Do I need to input the correct concentration to get the correct KD?

A: Yes, KD is calculated as KD = kd / ka. The kd is not dependent on concentration, but the ka is dependent on concentration because ka = (kobs – kd) / concentration. Therefore, if the concentration is entered incorrectly in the Analysis table, the ka and KD will be affected.

Q: What is being reported in the Agile R100 Analysis table and how is it relevant to my interaction?

A: On an Agile R100, the interaction is between the immobilized target and the analyte in solution, and the magnitude of sensor response (S), the observed binding rate (kobs), the association binding rate (ka), the dissociation binding rate (kd), and the dissociation constant (KD) are reported in the Analysis table.

For kinetic binding data, the KD, ka, and kd are needed. Agile R100 also reports the S and the kobs because the S is used to generate a dose response curve if multiple concentrations of analyte are tested, and the kobs is needed to calculate the ka (and therefore KD) of an interaction.

The magnitude of sensor response (S) and the observed binding rate (kobs) are first calculated during association (i.e. adding analyte in dilution buffer), and then the kd is calculated during dissociation (i.e. rinsing with dilution buffer). Once the S, kobs, and kd have been calculated, the ka and KD can also be reported.

Q: Why do I need to perform a dissociation step to calculate my KD?

A: During association, the observed binding is the sum of analytes associating and dissociating from the target as analytes dynamically come on and off the target. Therefore, the observed binding rate (kobs) term is needed to decouple the association binding rate (ka) and dissociation binding rate (kd).

During association, the kobs is recorded and calculated, and the kobs is different than the ka. Although ka depends on the concentration of the analyte, the ka is a constant value specific to an interaction and is calculated as ka = (kobs – kd) / concentration. As the analyte concentration varies, the kobs also varies so that the ka value is maintained for the specific interaction. While the ka will be a constant value for an interaction, the kobs will be different for every concentration tested. The kobs is necessary to determine ka but should not be used to compare interactions because of the dependency on concentration.

Once kobs has been calculated, the kd is simply be calculated when only dissociation is occurring (i.e. adding dilution buffer without analyte). Once the kd is calculated (and kobs are concentration are known), the ka can be decoupled from the kobs. KD can then be calculated as KD = kd / ka.

Hardware

Q: What is the VHD Cable and how do I use it?

A: The Agile R100 cartridge can connect directly to the reader, and the very high density (VHD) cable accessory provides the option of connecting the cartridge either directly to the reader or connecting using the VHD cable. If you have the VHD cable accessory, you have the additional option of using the cartridge in a wide range of experimental settings (e.g. with a temperature controlled incubator or in a biosafety cabinet) that may otherwise damage the reader. The cartridge and VHD cable can be separated from the reader post-experiment for easy decontamination with standard protocols.

Q: What are the dimensions of the system?

A: The Agile cartridge is approximately (L x W x H) 3.2” x 3” x 1.4”, and the AGILE reader is approximately 5.5” x 3” x 1.4”.

Q: What is the power supply of Agile R100?

A: Agile R100 connects to a computer via a USB to micro-USB cable and is powered by the computer. Agile R100 requires minimal processing power, and a laptop or computer running other applications can power Agile R100.