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General

Q: What reagents are supplied by Nanomedical Diagnostics?

A: The necessary reagents specific to each biosensor chip are provided with your biosensor chip order. For the COOH and NHS biosensor chips, Quench 1 and Quench 2 are included. For the NTA biosensor chips, NiCl2 is provided. For the NH2 biosensor chips, Quench 2 is included.

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

A: For all biosensor chips, you will need the buffers specific to your target and analyte. To immobilize target on the COOH, NHS, and NH2 biosensor chips, you will need biotechnology grade 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.0, 50 mM and culture or molecular grade 1X phosphate buffered saline (PBS) pH 7.4. For the COOH biosensor chips, you will need N-Hydroxysulfosuccinimide (sulfo-NHS; CAS No. 106627‑54‑7) and ultra High Purity (UHP) grade N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC; CAS No. 25952-53-8). For the NTA biosensor chips, you will need culture or molecular grade deionized water (CAS No. 7732-18-5).

Q: How do I learn to use Agile R100?

A: The Agile R100 Getting Started Kit (GSK) is a self-training package that can be run in 4 hours from the comfort of your bench, and it comes with every Agile R100 system. The GSK walks you through a positive control interaction (TNFa and SPD304), teaching you how to use the system and understand the analysis provided by Agile Plus software. Additional GSKs can be ordered separately if needed. Agile R100 also comes with an extensive User Manual for reference, and Field Application Scientists are on hand to provide special training to any number of users upon request. 

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

A: Field Applications 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: There are 5 types of biosensor chips to covalently or non-covalently link your target to the graphene surface: COOH, NHS, NH2, NTA, and FLEX. The COOH biosensor chips come with exposed carboxyl groups to covalently link target directly to the graphene surface using 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, NHS biosensor chips negate the need for EDC/sNHS while forming a covalent amide bond to a free amine group in your target. The NH2 biosensor chips have amine groups functionalized to the graphene surface, and EDC/sNHS activate the carboxyl groups on the target to react with the amine groups on the biosensor chip surface, forming a covalent amide bond.

His-tag proteins can be non-covalently immobilized to NTA biosensor chips, and the FLEX biosensor chips immobilize the target using hydrophobic interactions, noncovalent binding, or covalent binding, depending on the target and linking chemistry. These various binding chemistries make Agile R100 flexible and adaptable to established research and development pipelines.

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 Plus software, is the data still being recorded?

A: Yes, when you choose transistors in the Agile Plus 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 = koff / kon. The koff is not dependent on concentration, but the kon is dependent on concentration because kon = (kobs – koff) / concentration. Therefore, if the concentration is entered incorrectly in the Analysis table, the kon 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 rate (kobs), the association rate (kon), the dissociation rate (koff), and the dissociation constant (KD) are reported in the Analysis table.

For kinetic binding data, the KD, kon, and koff 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 kon (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 assay buffer), and then the koff is calculated during dissociation (i.e. rinsing with assay buffer). Once the S, kobs, and koff have been calculated, the kon 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 rate (kobs) term is needed to decouple the association rate (kon) and dissociation rate (koff).

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

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

Hardware

Q: What is the High Density Cable Accessory and how do I use it?

A: The Agile R100 cartridge connects directly to the reader for compact use. The cartridge can also attach to the reader via a specially-designed one-meter-long high density cable, which is an optional accessory. This enables additional use of the cartridge in a wide range of experimental settings (ex: 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 cartridge is approximately (L x W x H) 3.2” x 3” x 1.4”, and the 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.