Online protein activity detection device, SPR detector and use method thereof

Abstract

The invention relates to an online protein activity detection device, an SPR (Surface Plasmon Resonance) detector and a use method thereof, and belongs to the technical field of detection devices. The device comprises a first liquid storage ring which is provided with a first sample switching valve; the second liquid storage ring is provided with a second sample switching valve; the detector is provided with a channel switching valve, and a reference channel and an experiment channel which are communicated with the channel switching valve; one end of the dilution pipeline is communicated with the first liquid storage ring and the second liquid storage ring, and the other end is communicated with the detector; the buffer solution pipeline is respectively communicated with the first liquid storage ring and the second liquid storage ring. Through cooperative work of the sample switching valve, the channel switching valve, the dilution pipeline and the buffer solution pipeline, sample dilution, sample injection, detection and residual sample collection can be automatically completed, manual step-by-step treatment is not needed, the efficiency is remarkably improved, and the operation complexity is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of detection devices, and in particular to an online protein activity detection device based on the SPR principle, an SPR detector comprising the online protein activity detection device based on the SPR principle, and a method for using the online protein activity detection device based on the SPR principle. Background Art

[0002] In the study of complex biological samples, it is generally necessary to use techniques such as liquid chromatography and protein chromatography to separate the components of the complex sample, collect the individual components, and then use techniques such as SPR to screen and test the individual components until a limited number of components are screened. However, the component screening process is large in number. After the components are collected, they need to be manually processed and diluted individually before subsequent screening, which is time-consuming and labor-intensive. Summary of the Invention

[0003] In view of this, in order to solve the technical problem that the screening and separation of components in the prior art require manual processing of diluted samples separately and then subsequent screening work, which is time-consuming and labor-intensive, the first aspect of the present invention provides an online protein activity detection device based on the SPR principle. Through the coordinated work between the sample switching valve, the channel switching valve, the dilution pipeline and the buffer pipeline, it can automatically complete sample dilution, injection, detection and residual sample collection without manual step-by-step processing, significantly improving efficiency and reducing operation complexity.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] An online protein activity detection device based on the SPR principle, comprising:

[0006] A first liquid storage ring having a first sample switching valve;

[0007] A second liquid storage ring having a second sample switching valve;

[0008] A detector comprising a channel switching valve, a reference channel and an experimental channel connected to the channel switching valve;

[0009] a dilution line, one end of which is connected to the first liquid storage ring and the second liquid storage ring respectively, and the other end of which is connected to the detector;

[0010] The buffer solution pipeline is connected to the first liquid storage ring and the second liquid storage ring respectively.

[0011] Preferably, the first sample switching valve and the second sample switching valve are both six-way valves.

[0012] Preferably, the channel switching valve is a three-way valve.

[0013] Preferably, a first buffer pump is provided on the pipeline connecting the buffer pipeline and the first liquid storage ring;

[0014] A second buffer pump is provided on the pipeline communicating with the buffer pipeline and the second liquid storage ring.

[0015] In a second aspect, the present invention provides an SPR detector, comprising the above-mentioned online protein activity detection device based on the SPR principle.

[0016] In a third aspect, the present invention further provides a method for using the above-mentioned online protein activity detection device based on the SPR principle, comprising the following steps:

[0017] Step (1), chip loading: the chip is placed in the warehouse, the buffer solution is injected into the buffer solution pipeline at the same time, and all bubbles in the buffer solution pipeline are discharged;

[0018] Step (2), ligand immobilization: the chip is installed in the detector, the experimental channel is immobilized with the ligand, and the reference channel is a blank channel;

[0019] Step (21), chip cleaning: switch the second sample switching valve to the Load state, inject the cleaning liquid into the second liquid storage ring, switch the second sample switching valve to the Inject state, switch the channel switching valve to the reference and experimental channels in series, push the cleaning liquid into the liquid path of the detector, and after cleaning the chip, continue to inject the liquid to push away the cleaning liquid so that the buffer solution fills the pipeline;

[0020] Step (22), chip activation: switch the second sample switching valve to the Load state, inject the activation reagent into the second liquid storage ring, switch the second sample switching valve to the Inject state, switch the channel switching valve to the reference channel and the experimental channel in series, push the activation reagent into the liquid path of the detector, and after the chip is activated, continue to inject liquid to push away the activation reagent so that the buffer solution fills the pipeline;

[0021] Step (23), coupling: switch the second sample switching valve to the Load state, inject the ligand into the second liquid storage ring, switch the sample switching valve 2 to the Inject state, switch the channel switching valve to the experimental channel, and use pump 2 to push the ligand into the detector liquid path. After coupling, continue to inject liquid to push the ligand away, so that the buffer solution fills the pipeline and completes the coupling;

[0022] Step (24), closing: switch the second sample switching valve to the Load state, inject the blocking reagent into the second liquid storage ring, switch the second sample switching valve to the Inject state, switch the channel switching valve to the reference and experimental channels in series, push the blocking reagent into the liquid path of the detector, and after the chip is closed, continue to inject liquid to push away the blocking reagent so that the buffer solution fills the pipeline;

[0023] Step (3), sample detection: the front-end instrument discharges the separated components into the detection device for detection;

[0024] Step (31), the first sample switching valve is initially in the Load state;

[0025] Step (32), front-end instrument separation and sample injection: the front-end instrument is set to discharge the sample and push it into the first liquid storage ring of the first sample switching valve;

[0026] Step (33), the front-end instrument sends a trigger signal: after the front-end instrument pushes the sample into the first liquid storage ring, the trigger signal is sent to the SPR detector through the signal line;

[0027] Step (34), switching of the first sample switching valve: the SPR detector receives a trigger signal, switches the first sample switching valve to the Inject state, and prepares for sample detection. At the same time, the SPR curve in the software is reset and the signal is collected again;

[0028] Step (35), collection of remaining samples: after the first sample switching valve is switched to the Inject state, the front-end instrument can push the remaining sample into the collection device for sample collection;

[0029] Step (36), sample dilution: pushing the sample into the dilution line, and simultaneously filling the buffer line with liquid, using the buffer to dilute the sample in the dilution line;

[0030] Step (37), sample detection: the channel switching valve is switched to the reference channel and the experimental channel in series, pushing the diluted sample into the chip in the detector, and performing real-time signal acquisition at the same time.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] Fully automated online testing

[0033] Solve the problem of manual intervention: Through the coordinated work between the sample switching valve, channel switching valve, dilution pipeline and buffer pipeline, sample dilution, injection, detection and residual sample collection can be completed automatically without manual step-by-step processing, significantly improving efficiency and reducing operational complexity.

[0034] Real-time dynamic analysis

[0035] Shorten the detection cycle: Directly connect with front-end instruments such as liquid chromatography to intercept the separated components in real time and immediately dilute and test them, avoiding the delays of sample collection and manual transfer in traditional methods and improving the timeliness of experiments.

[0036] Adaptability to complex liquid environments

[0037] Intelligent dilution adjustment: Through the design of buffer lines and dilution lines, samples with high salt, acid, base or high organic solvent content can be adjusted to conditions suitable for SPR detection, expanding the scope of application of detection scenarios.

[0038] Dual-channel detection optimizes accuracy

[0039] Reference channel eliminates interference: The series connection or independent switching of the experimental channel and the reference channel (through the channel switching valve) can synchronously obtain the background signal, effectively eliminate environmental interference, and improve the accuracy and reliability of the test data.

[0040] Efficient sample recovery

[0041] Non-destructive collection of remaining samples: The tested samples enter the collection device directly through an independent pipeline, retaining unused components for subsequent verification or retesting, reducing sample waste.

[0042] System stability and flexibility: The cooperation between the liquid storage ring and the sample switching valve enables segmented sample interception and precise quantification;

[0043] Process scalability

[0044] The method for using the online protein activity detection device based on the SPR principle provided by the present invention is compatible with a variety of chips and ligands. Through an adjustable ligand immobilization process (such as activation, coupling, and blocking steps), it is adapted to different chip types (such as CM5 chips) and biomolecules (proteins, antibodies, etc.), enhancing the versatility of the device. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the overall main view of the SPR detector;

[0046] Figure 2 This is an overall schematic diagram of the online protein activity detection device based on the SPR principle provided by the present invention;

[0047] Figure 3 This is the Load state diagram of the first sample switching valve;

[0048] Figure 4 This is the Inject state diagram of the first sample switching valve;

[0049] Figure 5 This is the Load state diagram of the second sample switching valve;

[0050] Figure 6 This is the Inject state diagram of the first sample switching valve;

[0051] Figure 7 is a schematic diagram of the detector;

[0052] In the figure, 1, first liquid storage ring; 11, first sample switching valve; 2, second liquid storage ring; 21, second sample switching valve; 3, detector; 31, channel switching valve; 32, reference channel; 33, experimental channel; 4, dilution line; 5, buffer solution line. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative work shall fall within the scope of protection of the present invention.

[0054] In the description of the present invention, it should be noted that the terms "upper," "lower," "inner," "outer," "top / bottom," and the like, indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0055] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "installed," "provided with," "mounted / connected," and "connected" should be understood in a broad sense. For example, "connected" can mean a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be internal communication between two components. Those skilled in the art will be able to understand the specific meanings of the above terms in the present invention in specific circumstances.

[0056] like Figure 2-7 As shown, the present invention provides an online protein activity detection device based on the SPR principle, comprising:

[0057] The first liquid storage ring 1 has a first sample switching valve 11. The first sample switching valve 11 is a six-way valve having a front sample access port, a sample collection access port, a dilution line access port, and a buffer line access port. The following injection methods can be performed through the first sample switching valve 11, as follows:

[0058] The liquid discharged from the front-end instrument (front-end sample) is passed into the first liquid storage ring 1 through the first sample switching valve 11, and then to the collection end (such as Figure 3 As shown), after the sample fills the first liquid storage ring, the front-end instrument sends a signal to switch the first sample switching valve 11 to the internal liquid path (as shown Figure 4 As shown), the first liquid storage ring 1 is connected to the buffer line 5 and the dilution line 4, and the sample in the first liquid storage ring 1 is pushed into the dilution line 4. The sample in the first liquid storage ring 1 flows to the detector 3 (containing a chip) through the dilution line 4, and the remaining front-end samples are passed into the sample collection access end for collection.

[0059] The second liquid storage ring 2 has a second sample switching valve 21. The second sample switching valve 21 is a six-way valve having a sample injection access port, a waste liquid collection port, a dilution line access port, and a buffer line access port. The following injection methods can be performed through the second sample switching valve 21, as follows:

[0060] The second sample switching valve 21 is in the Load state (the sample is in the sample loading state, the sample enters the second liquid storage ring 2, and the excess sample is discharged and collected through the waste liquid collection end.) (such as Figure 5 As described above, the second liquid storage ring 2 is connected to the sample injection port and the waste liquid, and the sample is injected into the second liquid storage ring 2 using a syringe; then the second sample switching valve 21 is switched to the Inject state (injection state, the sample in the second liquid storage ring 2 is flushed into the detector by the mobile phase) (as Figure 6 As shown), the second liquid storage ring 2 is connected to the buffer line 5 and the dilution line 4, and the sample in the second liquid storage ring 2 is pushed into the dilution line 4, and the sample in the second liquid storage ring 2 flows to the detector 3 (chip) through the dilution line 4.

[0061] like Figure 7 As shown, the detector 3 has a channel switching valve 31, a reference channel 32 and an experimental channel 33 connected to the channel switching valve 31. The channel switching valve 31 has an experimental channel 33 and a reference channel 32. There are two ways for the solution to flow during the entire experiment, namely, flowing only through the experimental channel 33, and first flowing through the reference channel 32 and then through the experimental channel 33. The passage above the channel switching valve 31 is a common end, which is connected to the left or right flow path. When the channel switching valve is switched to the left, the liquid only flows through the experimental channel 33. When the channel switching valve 31 is switched to the right, the liquid first flows through the reference channel 32 and then through the experimental channel 33.

[0062] The channel switching valve 31 can be switched according to actual needs to achieve flexible conversion between the reference channel 32 and the experimental channel 33. The reference channel 32 is mainly used to provide a reference signal, which is used as a benchmark to calibrate and compare the detection signal in the experimental channel 33, thereby ensuring the accuracy and reliability of the test results. The experimental channel 33 is used to receive samples transported through the dilution line 4 and allow the sample to interact with the SPR sensor element on the chip to detect the protein activity of the sample. Through the precise control of the channel switching valve 31, efficient and continuous detection of different samples can be achieved, further improving the flexibility and automation of the entire detection process.

[0063] The dilution line 4 is connected to the first liquid storage ring 1 and the second liquid storage ring 2 at one end, and connected to the detector 3 at the other end. The design of the dilution line 4 not only realizes the flexible switching and dilution of samples, but also enhances the scalability of the entire detection process. Specifically, the dilution line 4 can be easily expanded to more sample processing channels by adding branch lines or connecting multiple liquid storage rings. This design enables multiple different samples to be processed and analyzed simultaneously within the same detection cycle, greatly improving the detection efficiency. In addition, the material selection of the dilution line 4 also fully considers chemical compatibility and durability, ensuring stable performance under various experimental conditions, thereby further improving the reliability and service life of the entire detection system.

[0064] The buffer line 5 is connected to the first liquid storage ring 1 and the second liquid storage ring 2 respectively. The design of the buffer line 5 also reflects the careful consideration of the scalability of the process. It not only provides a stable supply of buffer for the first liquid storage ring 1 and the second liquid storage ring 2, but also allows the type and concentration of the buffer to be flexibly adjusted according to experimental requirements. This design ensures that under different experimental conditions, a suitable liquid environment can be provided for the SPR sensor element, thereby maintaining the stability and accuracy of the detection. In addition, the flexible layout of the buffer line 5 also reserves space for possible system upgrades in the future, so that the entire online protein activity detection device can continue to evolve with the development of scientific research needs, and always maintain its advanced nature and practicality.

[0065] In the present invention, both the first sample switching valve 11 and the second sample switching valve 21 are six-way valves.

[0066] In the present invention, the channel switching valve 31 is a three-way valve.

[0067] In the present invention, a first buffer pump is provided on the pipeline connecting the buffer pipeline 5 and the first liquid storage ring 1;

[0068] A second buffer pump is provided on the pipeline connecting the buffer pipeline 5 and the second liquid storage ring 2 .

[0069] The design of these two buffer pumps not only ensures stable and continuous delivery of buffer to the first and second liquid storage rings, but also enables precise regulation of the buffer flow rate by precisely controlling the operating status of the pumps. This design greatly enhances the flexibility and controllability of experiments, allowing researchers to quickly adjust the buffer supply status according to different experimental requirements, thereby further optimizing experimental conditions and improving detection efficiency and accuracy. In addition, the introduction of the buffer pumps also opens up more possibilities for future system upgrades and functional expansion, allowing the entire online protein activity detection device to better adapt to the changes and developments in scientific research needs.

[0070] like Figure 1 As shown, in a second aspect, the present invention provides an SPR detector, comprising the above-mentioned online protein activity detection device based on the SPR principle. The SPR detector inherits all the advantages of the online protein activity detection device, such as high precision, high sensitivity, and process controllability and flexibility.

[0071] In a third aspect, the present invention further provides a method for using the above-mentioned online protein activity detection device based on the SPR principle, comprising the following steps:

[0072] Step (1), chip loading: the chip is placed in the warehouse, and the buffer solution is injected into the buffer solution pipeline 5 at the same time, and all bubbles in the buffer solution pipeline 5 are discharged;

[0073] Step (2), ligand immobilization: the chip is installed in the detector 3, the experimental channel 33 is immobilized with the ligand, and the reference channel 32 is a blank channel;

[0074] Step (21), chip cleaning: switch the second sample switching valve 21 to the Load state, inject the cleaning liquid into the second liquid storage ring 2, switch the second sample switching valve 21 to the Inject state, switch the channel switching valve 31 to the reference and experimental channels 33 in series, push the cleaning liquid into the liquid path of the detector 3, and after cleaning the chip, continue to inject liquid to push away the cleaning liquid so that the buffer solution fills the pipeline;

[0075] Step (22), chip activation: switch the second sample switching valve 21 to the Load state, inject the activation reagent into the second liquid storage ring 2, switch the second sample switching valve 21 to the Inject state, switch the channel switching valve 31 to the reference and experimental channels 33 in series, push the activation reagent into the liquid path of the detector 3, and after the chip is activated, continue to inject liquid to push away the activation reagent so that the buffer solution fills the pipeline;

[0076] Step (23), coupling: switch the second sample switching valve 21 to the Load state, inject the ligand into the second liquid storage ring 2, switch the sample switching valve 2 to the Inject state, switch the channel switching valve 31 to the experimental channel 33, and use the pump 2 to push the ligand into the detector 3 liquid path. After coupling, continue to inject liquid to push the ligand away, so that the buffer solution fills the pipeline, and the coupling is completed;

[0077] Step (24), closing: switch the second sample switching valve 21 to the Load state, inject the blocking reagent into the second liquid storage ring 2, switch the second sample switching valve 21 to the Inject state, switch the channel switching valve 31 to the reference and experimental channels 33 in series, push the blocking reagent into the liquid path of the detector 3, and after the chip is closed, continue to inject liquid to push away the blocking reagent so that the buffer solution fills the pipeline;

[0078] Step (3), sample detection: the front-end instrument discharges the separated components into the detection device for detection;

[0079] Step (31), the first sample switching valve 11 is initially in the Load state;

[0080] Step (32), front-end instrument separation and sample injection: the front-end instrument is set to discharge the sample and push it into the first liquid storage ring 1 of the first sample switching valve 11;

[0081] Step (33), the front-end instrument sends a trigger signal: after the front-end instrument pushes the sample into the first liquid storage ring 1, the trigger signal is sent to the SPR detector through the signal line;

[0082] Step (34), switching of the first sample switching valve 11: the SPR detector receives a trigger signal, switches the first sample switching valve 11 to the Inject state, and prepares for sample detection. At the same time, the SPR curve in the software is reset and the signal is collected again;

[0083] Step (35), collection of remaining samples: after the first sample switching valve 11 is switched to the Inject state, the front-end instrument can push the remaining sample into the collection device for sample collection;

[0084] Step (36), sample dilution: pushing the sample into the dilution line 4, while filling the buffer line 5 with liquid, using the buffer to dilute the sample in the dilution line 4;

[0085] Step (37), sample detection: the channel switching valve 31 is switched to the reference channel and the experimental channel 33 is connected in series, pushing the diluted sample to flow onto the chip in the detector 3, and performing real-time signal acquisition at the same time.

[0086] The following steps may also be included:

[0087] Step (4), data analysis: After the detection is completed, the SPR curve data is collected and saved, and the SPR curve is fitted and analyzed using software to obtain key parameters such as the sample's association rate constant, dissociation rate constant, and affinity, thereby evaluating the protein activity;

[0088] Step (5), system cleaning and resetting: After the sample test is completed, the second sample switching valve 21 is switched to the Load state, and the cleaning fluid is injected again to clean the system to ensure that there is no residue in the pipeline. Then, each valve and pump are reset to the initial state for next use.

[0089] In the above-mentioned usage method provided by the present invention, the entire usage process is reasonably designed, easy to operate, and can realize automated online detection, which greatly improves the efficiency and accuracy of protein activity detection. It also has good process scalability and can be flexibly adjusted and optimized according to actual needs.

[0090] The above description is only a preferred specific embodiment of the present invention; however, the protection scope of the present invention is not limited thereto; any technician familiar with the technical field within the technical scope disclosed by the present invention; any equivalent replacement or change based on the technical solution and improved conception of the present invention shall be covered within the protection scope of the present invention.

Claims

1. An online protein activity detection device based on the SPR principle, characterized in that: include: A first liquid storage ring having a first sample switching valve; A second liquid storage ring having a second sample switching valve; A detector comprising a channel switching valve, a reference channel and an experimental channel connected to the channel switching valve; a dilution line, one end of which is connected to the first liquid storage ring and the second liquid storage ring respectively, and the other end of which is connected to the detector; The buffer solution pipeline is connected to the first liquid storage ring and the second liquid storage ring respectively.

2. The online protein activity detection device based on the SPR principle according to claim 1, characterized in that: The first sample switching valve and the second sample switching valve are both six-way valves.

3. The online protein activity detection device based on the SPR principle according to claim 1, characterized in that: The channel switching valve is a three-way valve.

4. An online protein activity detection device based on the SPR principle according to any one of claims 1 to 3, characterized in that: A first buffer pump is provided on the pipeline communicating between the buffer pipeline and the first liquid storage ring; A second buffer pump is provided on the pipeline communicating with the buffer pipeline and the second liquid storage ring.

5. An SPR detector, characterized in that An online protein activity detection device based on the SPR principle according to any one of claims 1 to 4.

6. A method for using an online protein activity detection device based on the SPR principle according to any one of claims 1 to 4, characterized in that: The steps include: Step (1), chip loading: the chip is placed in the warehouse, the buffer solution is injected into the buffer solution pipeline at the same time, and all bubbles in the buffer solution pipeline are discharged; Step (2), ligand immobilization: the chip is installed in the detector, the experimental channel is immobilized with the ligand, and the reference channel is a blank channel; Step (21), chip cleaning: switch the second sample switching valve to the Load state, inject the cleaning liquid into the second liquid storage ring, switch the second sample switching valve to the Inject state, switch the channel switching valve to the reference and experimental channels in series, push the cleaning liquid into the liquid path of the detector, and after cleaning the chip, continue to inject the liquid to push away the cleaning liquid so that the buffer solution fills the pipeline; Step (22), chip activation: switch the second sample switching valve to the Load state, inject the activation reagent into the second liquid storage ring, switch the second sample switching valve to the Inject state, switch the channel switching valve to the reference channel and the experimental channel in series, push the activation reagent into the liquid path of the detector, and after the chip is activated, continue to inject liquid to push away the activation reagent so that the buffer solution fills the pipeline; Step (23), coupling: switch the second sample switching valve to the Load state, inject the ligand into the second liquid storage ring, switch the sample switching valve 2 to the Inject state, switch the channel switching valve to the experimental channel, and use pump 2 to push the ligand into the detector liquid path. After coupling, continue to inject liquid to push the ligand away, so that the buffer solution fills the pipeline and completes the coupling; Step (24), closing: switch the second sample switching valve to the Load state, inject the blocking reagent into the second liquid storage ring, switch the second sample switching valve to the Inject state, switch the channel switching valve to the reference and experimental channels in series, push the blocking reagent into the liquid path of the detector, and after the chip is closed, continue to inject liquid to push away the blocking reagent so that the buffer solution fills the pipeline; Step (3), sample detection: the front-end instrument discharges the separated components into the detection device for detection; Step (31), the first sample switching valve is initially in the Load state; Step (32), front-end instrument separation and sample injection: the front-end instrument is set to discharge the sample and push it into the first liquid storage ring of the first sample switching valve; Step (33), the front-end instrument sends a trigger signal: after the front-end instrument pushes the sample into the first liquid storage ring, the trigger signal is sent to the SPR detector through the signal line; Step (34), switching of the first sample switching valve: the SPR detector receives a trigger signal, switches the first sample switching valve to the Inject state, and prepares for sample detection. At the same time, the SPR curve in the software is reset and the signal is collected again; Step (35), collection of remaining samples: after the first sample switching valve is switched to the Inject state, the front-end instrument can push the remaining sample into the collection device for sample collection; Step (36), sample dilution: pushing the sample into the dilution line, and simultaneously filling the buffer line with liquid, using the buffer to dilute the sample in the dilution line; Step (37), sample detection: the channel switching valve is switched to the reference channel and the experimental channel in series, pushing the diluted sample into the chip in the detector, and performing real-time signal acquisition at the same time.

Images