Application of surface plasmon resonance technology in protein-protein interaction
Application areas of binding specificity, antibody selection, antibody quality control, disease mechanisms, drug discovery, biological therapy, biological treatment, biomarkers, ligand fishing, gene regulation, cell signaling, affinity chromatography, structure-function relationships, Small molecule interactions, etc.
Detection Principle Surface plasmon resonance (SPR) is an optical phenomenon that can be used to track biomolecular interactions in nature in real time. This method does not cause any damage to biomolecules and does not require any markers.
A biomolecule (target molecule) is first bonded to the surface of the biosensor, and a solution containing another biomolecule (analyte) capable of interacting with the target molecule is injected and flows through the surface of the biosensor. The combination of biomolecules causes an increase in the surface quality of the biosensor, causing the refractive index to increase in the same proportion, and changes in the biomolecular reaction are observed. This reaction is measured in units of reaction (RU): 1 RU = 1 pg protein/mm2 = 1 x 10-6 RIU (refractive index unit).
During the injection, the analyte forms a complex with the target molecule by convection and diffusion through the interaction surface, resulting in a change in analyte concentration. The application of nL orders of magnitude flow channels in microfluidic systems minimizes this change in concentration to ensure a high Mass Transfer Coefficient (km). To ensure that the mass transfer of the analyte is not limited, the concentration of target molecules bound to the surface of the biosensor must be low. When the analyte is injected, the analyte-target molecule complex forms on the surface of the biosensor, resulting in enhanced reaction. When the analyte is injected, the analyte-target molecule complex dissociates, resulting in a weakening of the reaction. The kinetic constant can be determined by fitting this reaction curve through a combined interaction model. The non-specific binding and total refractive index shift equal effect can be removed by referring to the curve subtraction function.
Biosensor and microfluidic system
SensiQ's SPR biosensor utilizes the optical sensor design developed by Texas Instruments and the Kretschmann SPR geometry to create high sensitivity and optical stability. The biosensor is disposable and its carboxylated surface is suitable for a variety of optimized bonding protocols. Biosensors are quick to install and can be used in seconds, making them easy to use. Functionalized biosensors can continue to be used even after storage for a period of time.
SensiQ's dual channel nL flow cell design facilitates real-time reference curve subtraction and ensures high mass transfer of analytes on the interaction surface of biosensors. Minimizing dead volume and sample scattering is critical to the acquisition of real and quantitative kinetic and affinity data. The durable jet design is easy to service and significantly reduces downtime.
Surface Plasmon Resonance Technology Surface Plasmon Resonance (SPR) is an optical phenomenon that can be used to track biomolecular interactions in nature in real time. This method does not cause any damage to biomolecules and does not require any markers.
A biomolecule (target molecule) is first bonded to the surface of the biosensor, and a solution containing another biomolecule (analyte) capable of interacting with the target molecule is injected and flows through the surface of the biosensor. The combination of biomolecules causes an increase in the surface quality of the biosensor, causing the refractive index to increase in the same proportion, and changes in the biomolecular reaction are observed. This reaction is measured in units of reaction (RU): 1 RU = 1 pg protein/mm2 = 1 x 10-6 RIU (refractive index unit).
During the injection, the analyte forms a complex with the target molecule by convection and diffusion through the interaction surface, resulting in a change in analyte concentration. The application of nL orders of magnitude flow cells in a microfluidic system minimizes this change in concentration to ensure a high Mass Transfer Coefficient (km). To ensure that the mass transfer of the analyte is not limited, the concentration of target molecules bound to the surface of the biosensor must be low. When the analyte is injected, the analyte-target molecule complex forms on the surface of the biosensor, resulting in enhanced reaction. When the analyte is injected, the analyte-target molecule complex dissociates, resulting in a weakening of the reaction. The kinetic constant can be determined by fitting this reaction curve through a combined interaction model. The non-specific binding and total refractive index shift equalization effects can be removed by reference curve subtraction.
Experimental design of affinity pairs includes:
Protein – Protein (Protein–Protein)
Peptide–Receptor
Antibody – Antigen (Antibody–Antigen)
Membrane Receptor–Ligand
Lectin–Polysacharride/Glycoprotein
Protein–Small Molecule
Protein–Nucleic Acid
Cell-ligand (Cell–Ligand)
Any pair of affinity molecules, one (target molecule) is bound to the biosensor surface and the other (analyte) is placed in solution. The affinity complex is formed when the solution containing the analyte flows through the surface of the biosensor bonded to the target molecule.
The SensiQ is equipped with a dual channel flow injection analytical microfluidic system with an internal 85nL flow cell. SensiQ uses a disposable SPR biosensor for easy loading and unloading. The biosensor surface is coated with a single layer of Carboxylated Oligoethyleneoxide matrix to bond a variety of biomolecules and effectively block non-specific binding and denaturation. This method of binding the target biomolecule to both the surface of the solid phase biosensor and the liquid phase enhances the contact between the two affinity molecules while avoiding the kinetic analysis due to human factors. The complexity.
SensiQ has a variety of bonding options to improve the experimental design to support the attachment of biomolecules. The most common coupling method is amine coupling with EDC/NHS. Other bonding methods, such as maleimide-thiol, reductive amination, hydrazide-aldehyde, affinity capture, etc., may also be used. One of the SensiQ dual channel detections can be used to generate an appropriate reference curve. When the surface chemistry is fixed, add up to 250 μl of sample and a stable baseline is generated when the buffer flows through the biosensor surface. Sample injection and timing are done through automated control. With the Experiment Setup Wizard feature, users can record multiple injection cycles with high repeatability.
controlling software
SensiQ's control software acquires and displays data from both channels simultaneously and in real time as the original response curve is generated. The data in the reference channel is subtracted to compensate for thermal drift, non-specific binding, and total refractive index shift equalization effects, resulting in clear and high quality experimental data. The control software simply adds a reporting point to the reaction curve to determine the binding reaction produced after sample injection. The addition of reporting points can be done manually at any time during the experiment, or by a program preset in a fixed experimental period. All the reporting points listed in the table, together with the relevant event records, are available for investigation. Once the data file is saved, it can be reopened and edited by the control software.
SensiQ control software's simple and straightforward user interface makes experiment design and efficient and time-saving. The Experiment Wizard feature simplifies the setup process and improves experimental repeatability.
Analysis of the QDATTM analysis software response curve and subsequent calculations of kinetic and affinity data are often cumbersome and time consuming. SensiQ's QDATTM analysis software greatly simplifies the data analysis process and provides a simple, time-saving, and reliable means of measuring the dynamics and affinity of researchers.
QDATTM is the latest generation of analysis software developed on top of Biologic Software's widely used Clamp and Scrubber architecture to successfully analyze high quality data collected in minutes. QDATTM's concise and friendly interface guides users through a series of steps to complete the measurement of information in seconds. QDATTM's model fitting uses numerical integration and optimized curve fitting algorithms to quickly estimate the best fit parameter values, ensure the fit of the interaction model and data set to determine kinetic and affinity constants, and concentration analysis. QDATTM also provides a simple residual plot and residual standard deviation to quantitatively assess the degree of fit. The dynamics fitting model of QDATTM includes a Pseudo-first-order Binding Model and a Pseudo-first-order Binding Model with Mass Transport.
Technical parameters One-time biosensor is the number of flow cells 2
Flow cell selection 1, or 2, or 1 and 2
Flow cell area 2.2 mm2
Flow cell volume 85 nL (high mass transfer rate)
Sample added to manual (syringe)
Sample injection automatic computer control manual computer control sample dual channel simultaneous injection is sample injection volume 10–250 μL
Sample syringe pump built-in external sample flow rate 5–150 μL / min custom (< 250 μL / min)
Internal dead volume < 1.5 μL
The real-time reference curve subtraction is the refractive index range of 1.32–1.401.33–1.40
Short-term background noise < 0.25 RU< 1 RU
Long-term background noise < 0.30 RU/min (when the environment changes < 3 ° C / h)
Temperature control 15–40°C room temperature size (W x H x D) 35.0 x 34.2 x 38.8 cm22.9 x 15.2 x 27.9 cm
Weight 15.9 kg 3.6 kg
Power supply 100–240 V, 50/60 Hz
Working range lower molecular weight < 200 Da< 250 Da
Ka (binding rate constant) 1 x 107 M–1s–1
Kd (dissociation rate constant) 10–6–10–1 s–1
KD(kd / ka)10–4–10–10 M
Concentration < 10–10–10–3 M
Sensor surface chemistry
Amine coupling fixation (COOH1 and COOH2 chips)
Due to the ubiquity of amine groups, immobilization of ligands by amine coupling is suitable for most biomolecules. To date, we have found that this method randomly fixes ligands and usually yields high quality results. Therefore, it is not necessary to directly fix a specific site.
The most common method is to activate the carboxyl group using an aqueous mixture of NHS and EDC to produce an amine-based active lipid. This process has several benefits:
n No derivatization, no labeling, can fix most biomolecules
n produces a large number of stable covalent bonds to prevent the ligand from leaching from the surface
n is very effective in a wide range of pH values
n Biomolecules do not need to be exposed to harsh conditions
n It is easy to control the fixed conditions to prevent excessive cross-linking with the surface
n Preparation of chemical reagents, frozen for several months
Affinity capture of surface histidine tagged proteins (HisCap and HisHiCap chips)
ICx Nomadics' HisCap chip makes the immobilization of polyhistidine-tagged proteins stable and reversible, and makes surface plasmon resonance (SPR) experiments simpler. The baseline with immobilized proteins is very stable and can be used for kinetic analysis experiments.
HisCap chip:
n provides the most convenient means of directly immobilizing His-tagged proteins.
n can also be applied to any protein with a sufficient number of histidine residues.
The HisCap chip uses the NTA-Ni technology developed by Hoffman-LaRoche to attach proteins. In this technique, the side chain imidazole of histidine of the protein of interest cooperates with the surface-attached NTA-Ni complex as shown. This technique is very effective as long as the protein has sufficient histidine. A typical histidine tag is 6 histidines, but 3 are also possible.
HisCap chip advantages:
n His-tagging is a long-established standard technique in the laboratory for recombinant protein work.
n Capture of His-tagged proteins using the HisCap chip to produce a stable baseline.
n In mild conditions, the chip can be regenerated. For example EDTA or imidazole.
n Reusable HisCap chip.
Vesicle capture membrane receptor interaction (VesCap chip)
The ICX vesicle capture (VesCap) chip can be used to study the interaction of molecules with cell membranes and liposomes for real-time, label-free experiments. In the VesCap chip, the lipid bilayer appears to be in a natural cellular environment, and various cell membrane components in its own structure can be freely mixed in the true model of the cell membrane. We have not been able to confirm that the vesicles dissolve into the single membrane bilayer, but this is extremely possible on a two-dimensional surface.
n A good experimental model should contain drugs, toxins, and peripheral membrane-associated proteins involved in cellular signaling.
n Membrane proteins and associated proteins interact only in real cell membranes
n can immobilize the receptor/ligand of the liposome in the liposome
VesCap chip properties:
n The structure of the lipid bilayer and membrane protein remains unchanged. The vesicle capture at the sensor surface is non-covalent, allowing the membrane components in any direction to freely fuse.
The n PEG-n-ammonium layer exhibits a simple two-dimensional interaction plane.
n The preparation of vesicles and lipids attached to the surface is simple.
n VesCap chemistry is particularly suitable for a cell line that overexpresses surface receptors
The regeneration of the n VesCap chip is very simple. The combination of surfactant and solvent removes all of the vesicles on the surface of the VesCap chip, thereby regenerating the VesCap chip.
Avidin-biotin immobilization (BioCap and AvCap chips)
The immobilization of biomolecules by avidin-biotin-based methods is simple and effective, and is still widely welcomed by researchers today. Using this technology, the BioCap chip and the AvCap chip reliably immobilize the ligand. The schematic diagram depicts the two methods of fixation as follows. The main advantages are:
n does not depend on the isoelectric point of the protein
n requires only a small amount of ligand
n Commercially available, a wide range of biotin reagents
n Biotin kit is easy to operate
n fixation only requires a simple injection
n When the required Rmax is reached, stopping the injection can precisely control the concentration of the fixed conjugate
n has a very low electrostatic charge on the surface relative to the COOH chip
n A biotin reaction usually produces enough product to be immobilized in an unlimited amount.
Detection Principle Surface plasmon resonance (SPR) is an optical phenomenon that can be used to track biomolecular interactions in nature in real time. This method does not cause any damage to biomolecules and does not require any markers.
A biomolecule (target molecule) is first bonded to the surface of the biosensor, and a solution containing another biomolecule (analyte) capable of interacting with the target molecule is injected and flows through the surface of the biosensor. The combination of biomolecules causes an increase in the surface quality of the biosensor, causing the refractive index to increase in the same proportion, and changes in the biomolecular reaction are observed. This reaction is measured in units of reaction (RU): 1 RU = 1 pg protein/mm2 = 1 x 10-6 RIU (refractive index unit).
During the injection, the analyte forms a complex with the target molecule by convection and diffusion through the interaction surface, resulting in a change in analyte concentration. The application of nL orders of magnitude flow channels in microfluidic systems minimizes this change in concentration to ensure a high Mass Transfer Coefficient (km). To ensure that the mass transfer of the analyte is not limited, the concentration of target molecules bound to the surface of the biosensor must be low. When the analyte is injected, the analyte-target molecule complex forms on the surface of the biosensor, resulting in enhanced reaction. When the analyte is injected, the analyte-target molecule complex dissociates, resulting in a weakening of the reaction. The kinetic constant can be determined by fitting this reaction curve through a combined interaction model. The non-specific binding and total refractive index shift equal effect can be removed by referring to the curve subtraction function.
Biosensor and microfluidic system
SensiQ's SPR biosensor utilizes the optical sensor design developed by Texas Instruments and the Kretschmann SPR geometry to create high sensitivity and optical stability. The biosensor is disposable and its carboxylated surface is suitable for a variety of optimized bonding protocols. Biosensors are quick to install and can be used in seconds, making them easy to use. Functionalized biosensors can continue to be used even after storage for a period of time.
SensiQ's dual channel nL flow cell design facilitates real-time reference curve subtraction and ensures high mass transfer of analytes on the interaction surface of biosensors. Minimizing dead volume and sample scattering is critical to the acquisition of real and quantitative kinetic and affinity data. The durable jet design is easy to service and significantly reduces downtime.
Surface Plasmon Resonance Technology Surface Plasmon Resonance (SPR) is an optical phenomenon that can be used to track biomolecular interactions in nature in real time. This method does not cause any damage to biomolecules and does not require any markers.
A biomolecule (target molecule) is first bonded to the surface of the biosensor, and a solution containing another biomolecule (analyte) capable of interacting with the target molecule is injected and flows through the surface of the biosensor. The combination of biomolecules causes an increase in the surface quality of the biosensor, causing the refractive index to increase in the same proportion, and changes in the biomolecular reaction are observed. This reaction is measured in units of reaction (RU): 1 RU = 1 pg protein/mm2 = 1 x 10-6 RIU (refractive index unit).
During the injection, the analyte forms a complex with the target molecule by convection and diffusion through the interaction surface, resulting in a change in analyte concentration. The application of nL orders of magnitude flow cells in a microfluidic system minimizes this change in concentration to ensure a high Mass Transfer Coefficient (km). To ensure that the mass transfer of the analyte is not limited, the concentration of target molecules bound to the surface of the biosensor must be low. When the analyte is injected, the analyte-target molecule complex forms on the surface of the biosensor, resulting in enhanced reaction. When the analyte is injected, the analyte-target molecule complex dissociates, resulting in a weakening of the reaction. The kinetic constant can be determined by fitting this reaction curve through a combined interaction model. The non-specific binding and total refractive index shift equalization effects can be removed by reference curve subtraction.
Experimental design of affinity pairs includes:
Protein – Protein (Protein–Protein)
Peptide–Receptor
Antibody – Antigen (Antibody–Antigen)
Membrane Receptor–Ligand
Lectin–Polysacharride/Glycoprotein
Protein–Small Molecule
Protein–Nucleic Acid
Cell-ligand (Cell–Ligand)
Any pair of affinity molecules, one (target molecule) is bound to the biosensor surface and the other (analyte) is placed in solution. The affinity complex is formed when the solution containing the analyte flows through the surface of the biosensor bonded to the target molecule.
The SensiQ is equipped with a dual channel flow injection analytical microfluidic system with an internal 85nL flow cell. SensiQ uses a disposable SPR biosensor for easy loading and unloading. The biosensor surface is coated with a single layer of Carboxylated Oligoethyleneoxide matrix to bond a variety of biomolecules and effectively block non-specific binding and denaturation. This method of binding the target biomolecule to both the surface of the solid phase biosensor and the liquid phase enhances the contact between the two affinity molecules while avoiding the kinetic analysis due to human factors. The complexity.
SensiQ has a variety of bonding options to improve the experimental design to support the attachment of biomolecules. The most common coupling method is amine coupling with EDC/NHS. Other bonding methods, such as maleimide-thiol, reductive amination, hydrazide-aldehyde, affinity capture, etc., may also be used. One of the SensiQ dual channel detections can be used to generate an appropriate reference curve. When the surface chemistry is fixed, add up to 250 μl of sample and a stable baseline is generated when the buffer flows through the biosensor surface. Sample injection and timing are done through automated control. With the Experiment Setup Wizard feature, users can record multiple injection cycles with high repeatability.
controlling software
SensiQ's control software acquires and displays data from both channels simultaneously and in real time as the original response curve is generated. The data in the reference channel is subtracted to compensate for thermal drift, non-specific binding, and total refractive index shift equalization effects, resulting in clear and high quality experimental data. The control software simply adds a reporting point to the reaction curve to determine the binding reaction produced after sample injection. The addition of reporting points can be done manually at any time during the experiment, or by a program preset in a fixed experimental period. All the reporting points listed in the table, together with the relevant event records, are available for investigation. Once the data file is saved, it can be reopened and edited by the control software.
SensiQ control software's simple and straightforward user interface makes experiment design and efficient and time-saving. The Experiment Wizard feature simplifies the setup process and improves experimental repeatability.
Analysis of the QDATTM analysis software response curve and subsequent calculations of kinetic and affinity data are often cumbersome and time consuming. SensiQ's QDATTM analysis software greatly simplifies the data analysis process and provides a simple, time-saving, and reliable means of measuring the dynamics and affinity of researchers.
QDATTM is the latest generation of analysis software developed on top of Biologic Software's widely used Clamp and Scrubber architecture to successfully analyze high quality data collected in minutes. QDATTM's concise and friendly interface guides users through a series of steps to complete the measurement of information in seconds. QDATTM's model fitting uses numerical integration and optimized curve fitting algorithms to quickly estimate the best fit parameter values, ensure the fit of the interaction model and data set to determine kinetic and affinity constants, and concentration analysis. QDATTM also provides a simple residual plot and residual standard deviation to quantitatively assess the degree of fit. The dynamics fitting model of QDATTM includes a Pseudo-first-order Binding Model and a Pseudo-first-order Binding Model with Mass Transport.
Technical parameters One-time biosensor is the number of flow cells 2
Flow cell selection 1, or 2, or 1 and 2
Flow cell area 2.2 mm2
Flow cell volume 85 nL (high mass transfer rate)
Sample added to manual (syringe)
Sample injection automatic computer control manual computer control sample dual channel simultaneous injection is sample injection volume 10–250 μL
Sample syringe pump built-in external sample flow rate 5–150 μL / min custom (< 250 μL / min)
Internal dead volume < 1.5 μL
The real-time reference curve subtraction is the refractive index range of 1.32–1.401.33–1.40
Short-term background noise < 0.25 RU< 1 RU
Long-term background noise < 0.30 RU/min (when the environment changes < 3 ° C / h)
Temperature control 15–40°C room temperature size (W x H x D) 35.0 x 34.2 x 38.8 cm22.9 x 15.2 x 27.9 cm
Weight 15.9 kg 3.6 kg
Power supply 100–240 V, 50/60 Hz
Working range lower molecular weight < 200 Da< 250 Da
Ka (binding rate constant) 1 x 107 M–1s–1
Kd (dissociation rate constant) 10–6–10–1 s–1
KD(kd / ka)10–4–10–10 M
Concentration < 10–10–10–3 M
Sensor surface chemistry
Amine coupling fixation (COOH1 and COOH2 chips)
Due to the ubiquity of amine groups, immobilization of ligands by amine coupling is suitable for most biomolecules. To date, we have found that this method randomly fixes ligands and usually yields high quality results. Therefore, it is not necessary to directly fix a specific site.
The most common method is to activate the carboxyl group using an aqueous mixture of NHS and EDC to produce an amine-based active lipid. This process has several benefits:
n No derivatization, no labeling, can fix most biomolecules
n produces a large number of stable covalent bonds to prevent the ligand from leaching from the surface
n is very effective in a wide range of pH values
n Biomolecules do not need to be exposed to harsh conditions
n It is easy to control the fixed conditions to prevent excessive cross-linking with the surface
n Preparation of chemical reagents, frozen for several months
Affinity capture of surface histidine tagged proteins (HisCap and HisHiCap chips)
ICx Nomadics' HisCap chip makes the immobilization of polyhistidine-tagged proteins stable and reversible, and makes surface plasmon resonance (SPR) experiments simpler. The baseline with immobilized proteins is very stable and can be used for kinetic analysis experiments.
HisCap chip:
n provides the most convenient means of directly immobilizing His-tagged proteins.
n can also be applied to any protein with a sufficient number of histidine residues.
The HisCap chip uses the NTA-Ni technology developed by Hoffman-LaRoche to attach proteins. In this technique, the side chain imidazole of histidine of the protein of interest cooperates with the surface-attached NTA-Ni complex as shown. This technique is very effective as long as the protein has sufficient histidine. A typical histidine tag is 6 histidines, but 3 are also possible.
HisCap chip advantages:
n His-tagging is a long-established standard technique in the laboratory for recombinant protein work.
n Capture of His-tagged proteins using the HisCap chip to produce a stable baseline.
n In mild conditions, the chip can be regenerated. For example EDTA or imidazole.
n Reusable HisCap chip.
Vesicle capture membrane receptor interaction (VesCap chip)
The ICX vesicle capture (VesCap) chip can be used to study the interaction of molecules with cell membranes and liposomes for real-time, label-free experiments. In the VesCap chip, the lipid bilayer appears to be in a natural cellular environment, and various cell membrane components in its own structure can be freely mixed in the true model of the cell membrane. We have not been able to confirm that the vesicles dissolve into the single membrane bilayer, but this is extremely possible on a two-dimensional surface.
n A good experimental model should contain drugs, toxins, and peripheral membrane-associated proteins involved in cellular signaling.
n Membrane proteins and associated proteins interact only in real cell membranes
n can immobilize the receptor/ligand of the liposome in the liposome
VesCap chip properties:
n The structure of the lipid bilayer and membrane protein remains unchanged. The vesicle capture at the sensor surface is non-covalent, allowing the membrane components in any direction to freely fuse.
The n PEG-n-ammonium layer exhibits a simple two-dimensional interaction plane.
n The preparation of vesicles and lipids attached to the surface is simple.
n VesCap chemistry is particularly suitable for a cell line that overexpresses surface receptors
The regeneration of the n VesCap chip is very simple. The combination of surfactant and solvent removes all of the vesicles on the surface of the VesCap chip, thereby regenerating the VesCap chip.
Avidin-biotin immobilization (BioCap and AvCap chips)
The immobilization of biomolecules by avidin-biotin-based methods is simple and effective, and is still widely welcomed by researchers today. Using this technology, the BioCap chip and the AvCap chip reliably immobilize the ligand. The schematic diagram depicts the two methods of fixation as follows. The main advantages are:
n does not depend on the isoelectric point of the protein
n requires only a small amount of ligand
n Commercially available, a wide range of biotin reagents
n Biotin kit is easy to operate
n fixation only requires a simple injection
n When the required Rmax is reached, stopping the injection can precisely control the concentration of the fixed conjugate
n has a very low electrostatic charge on the surface relative to the COOH chip
n A biotin reaction usually produces enough product to be immobilized in an unlimited amount.
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