Syringe Test v1.2

You use the AssayMAP Syringe Test Kit on the AssayMAP Bravo Platform to verify the integrity of the probes and syringes in the Bravo 96AM Head. The test kit is designed to evaluate all the syringes in the head concurrently while the head is mounted on the Bravo Platform.

Description

When to use the AssayMAP Syringe Test Kit

•	As a part of routine hardware quality control to verify head functionality before conducting a run

•	To verify the functioning of one or more syringes that produce suspicious results

•	After a Bravo 96AM Head collision in which syringes may have been damaged

•	After replacing any syringes in the Bravo 96AM Head

How it works

You run a protocol that uses test cartridges mounted on the Bravo 96AM Head. The protocol quickly aspirates a volume of test solution, which creates a partial vacuum in the syringes. After an equilibration period, the aspirated test solution is dispensed into a microplate and the volumes of each well is calculated by measuring the absorbance of the test solution in a microplate reader.

During aspiration, damaged syringes will aspirate air and the resulting volume dispensed will be reduced. The affected syringes are then identified during the data analysis of the microplate reader results.

 

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AssayMAP Syringe Test Kit components

One AssayMAP Syringe Test Kit provides enough materials and reagents to perform 20 tests of the 96 syringes in the Bravo 96AM head.

The following table provides a description of the kit components.

 

Component	Description	Part Number	Quantity
Syringe Test Kit, which includes the following:	The shelf life of the Test solution is 6 months from date of purchase when stored at ambient temperature.	G5496-60050	1
• AssayMAP Resin-Free Cartridges	Empty cartridges used for testing	G5496-60009	1 rack
• Test solution	Aqueous colormetric solution with appropriate viscosity	5190-7471	3 bottle (250 mL each)
• AssayMAP Cartridge use and storage guide	Instructions on use and storage	G5496-90002C	1
• Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom	Required reusable labware	201254-100	2
• 96 Greiner 655101 PS Clr Rnd Well Flat Btm	Required reusable labware	655101	2

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Hardware requirements

AssayMAP Bravo Platform

 

 

The following figure and table show the components of the AssayMAP Bravo Platform, which are required for running the AssayMAP protocols.

Figure. AssayMAP Bravo Platform components

 

 

Item	Required hardware
1	Gripper upgrade
2	Bravo 96AM Head
3	96AM Wash Station or the later model 96 Channel Wash Station
4	Pump Module 2.0 and two carboys
5	96AM Cartridge & Tip Seating Station
6	Risers, 146 mm
7	STC controller
8	Peltier Thermal Station with custom plate nest
9	Thermal plate insert
10	Orbital Shaking Station with Control Unit

 

To avoid a hardware crash and equipment damage, ensure that the wash station contains the white wide-bore chimneys when using the 25 µL cartridges.

 

Note: The white wide-bore chimneys work for both 5-µL and 25-µL cartridges and are standard on wash stations purchased in 2020 onward. The wide-bore chimneys are white plastic, whereas the standard-bore chimneys are a semi-clear plastic. For details, see the 96 Channel Wash Station Maintenance Guide.

 

 

Additional required equipment

 

Required equipment	Description
Centrifuge	Centrifuge with a microplate rotor capable of achieving 500 x g
Microplate reader	Spectrophotometer capable of detecting absorbance at 425 nm in a 96-well microplate and generating a text (TXT) file of the data. Note: Although 425 nm is optimal, 405 nm is also acceptable.

 

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Software requirements

 

 

The following table lists the minimum software requirements:

 

Software	Version
Agilent VWorks Automation Control	13.1.5
Agilent Protein Sample Prep Workbench	3.2
Microsoft Excel Required for the reagent volume calculators and method setup tools.	2016

 

 

 

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Labware requirements

The Syringe Test requires the following labware:

•	Open reservoir, (2 per test) to hold water (deck location 4) and diluted test solution (deck location 7).

•	96-well microplate, (2 per test) for test sample collection and a centrifugation balance plate (deck locations 8 and 9).

The following figure shows the labware locations on the Bravo deck. The table lists the options for labware type by deck location.

 

Table Labware options by deck location

 

Labware options	Manufacturer part number	Deck location
Reservoir, Seahorse 201254-100, PP, no walls, pyramid bottom	Agilent 201254-100	4, 7 (default)
Reservoir, Axygen Scientific RES‑SW96-LP, 86mL pyramid bottom	Axygen Scientific RES‑SW96-LP	4, 7
12 Column, Low Profile Reservoir, Natural PP	Agilent 201280-100	4, 7
96 V11 Manual Fill Reservoir	Agilent G5498B#049	4, 7
96 Greiner 655101 PS Clr Rnd Well Flat Btm	Greiner Bio-One 655101	8, 9 (default)
96 Nunc 269620, Flat Bottom, Polystyrene	Thermo-Fisher 269620	8, 9
96 Corning 9017, Flat bottom, polystyrene, clear	Corning 9017	8, 9

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Workflow

 

Step	For this task...	See...
1	Run the Startup protocol in the System Startup/Shutdown utility.	Running the Startup protocol
2	Launch the Syringe Test utility.	Accessing the Syringe Test utility
3	Set up the Bravo Platform deck for the syringe test.	Setting up the test
4	Run the Syringe Test	Running the syringe test
5	Analyze the results	Analyzing the results
6	Verify defective syringes	Verifying defective syringes

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Accessing the Syringe Test utility

To access the Syringe Test utility:

Open the Utility Library, locate the Syringe Test utility, and then click Utility.

 

The following figure shows the Syringe Test utility interface.

 

 

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Setting up the test

The following procedure specifies volume requirements for the default labware selections. If you choose different labware types from the list of options, you should adjust the volumes accordingly.

To set up the test:

1	Place the accessories or components on the AssayMAP Bravo deck using the following figure and table as a guide.

 

 

Deck location	Accessory or component	Purpose
1	96AM Wash Station or the later model 96 Channel Wash Station	Washes syringes and exterior cartridges
2	96AM Cartridge & Tip Seating Station	Ensures proper seating of the cartridges on the head
4	Reservoir, Seahorse 201254-100, PP (default)*	Holds water for dilution of test solution.
6	Cartridge rack with 96 resin-free cartridges	Holds test cartridges at beginning and end of protocol
7	Reservoir, Seahorse 201254-100, PP (default)*	Holds the 1X test solution
8	96 Greiner 655101 PS Clr Rnd Well Flat Btm (default)*	Microplate of same type as the collection plate for balancing in the centrifuge
9	96 Greiner 655101 PS Clr Rnd Well Flat Btm (default)*	Receives the output from the syringes
* For other options, see Labware requirements.

2	Optional. Click Restore Defaults to set all Application Settings to their defaults.

3	In the Labware Table area, select the labware you are using for the protocol run.

4	Optional. To have the Centrifuge Balance Plate filled with 200 µL water by the Bravo Platform, select the Generate Balance Plate check box.

5	Dispense 80 mL of water into the reservoir on deck location 4.

6	Dispense 35 mL of the Test Solution into the reservoir on deck location 7.

 

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Running the syringe test

To run the syringe test:

1	Ensure that the labware positions match the Deck Layout area in the form, and that the labware are properly seated in the platepads.

 

 

Improperly seated labware can cause a hardware collision, resulting in equipment damage. Ensure that all labware are properly seated within the alignment features of their respective platepads.

 

2	Confirm that the selected labware in the Labware Table area is the labware you are using for the syringe test.

 

Incorrect labware selections can cause a hardware collision, resulting in equipment damage. Ensure that the selections in the Labware Table exactly match the physical labware present on the AssayMAP Bravo deck.

 

3	Click Run Syringe Test.

Refer to the following table for a summary of the automation movements that occur during the test execution.

 

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Automation movements during the syringe test run

 

Protocol step	Head moves to location...	Action
1	1	Washes the syringes at the wash station.
2	6	Mounts the test cartridges onto the head.
	2	Parks the cartridges at the 96AM Cartridge & Tip Seating Station.
3	4	Aspirates 100 µL from the Water Reservoir.
	9	Dispenses 100 µL water to the Collection Plate.
4 (Generate Balance Plate option only)	4	Aspirates 200 µL from the Water Reservoir.
	8	Dispenses 200 µL to the Centrifuge Balance Plate.
5	4	Aspirates water.
	6	Mounts cartridges.
	1	Primes cartridges by dispensing water at the wash station.
	2	Parks cartridges and aspirates air gap.
6	6	Mounts cartridges.
	7	Submerges cartridges in Test Solution. Rapidly aspirates 225 µL Test Solution and equilibrates for 5 min with the tips submerged
7	2	Parks cartridges at the 96AM Cartridge & Tip Seating Station.
8	1	Dispenses 125 µL
	9	Dispenses remaining liquid and mixes.
9	2	Parks cartridges at the 96AM Cartridge & Tip Seating Station.

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Analyzing the results

How the data is analyzed

The absorbance measurement of each well is compared to an expected reference value, which is determined from a statistical analysis of the data from all wells.

Any syringe that has a corresponding value > 10% of the reference value fails the test.

To analyze the syringe test results:

1	After the run is completed, remove the collection microplate and the balance microplate from the Bravo deck and spin them in a centrifuge at 500 x g for 1 min to remove any bubbles within the wells.

2	Set up the plate reader to:

•	Detect absorbance at 425 nm

Note: Although 425 nm is optimal, 405 nm is also acceptable.

•	Save the results to a file with a text (TXT) file format

Note: Alternatively, you can save the data to a Microsoft Excel file (XLSX) and then use the copy and paste commands to transfer the data to the Analysis Tool.

3	Transfer the file to the computer running the Protein Sample Prep Workbench.

4	In the AssayMAP Syringe Test: Using AssayMAP form, click Analysis Tool to open the AssayMAP Syringe Test Analysis Tool.

 

The AssayMAP Syringe Test Analysis Tool opens in Microsoft Excel.

 

5	Ensure that you enable content in Microsoft Excel.

6	Click Select data. In the Data Selection Options dialog box, click Import New Data.

 

7	In the Import Text File dialog box, select the text (TXT) data file you want to analyze and click Open.

8	Follow the instructions of the text import wizard:

a	In the Text Import Wizard Step 1 of 3 dialog box, click Finish to accept the default settings.

b	In the Import Data dialog box, make sure Existing worksheet is selected and then click OK.

 

When the Select Data dialog box appears asking you to Select a continuous 8 row by 12 column range of cells to analyze, select the 96 cells you want to analyze and click OK.

You must select 96 cells, otherwise you will receive a message requesting you to select 96 entries.

 

The data appears in the Analysis Tool window.

 

9	Click Analyze. The Analysis Tool analyzes the imported data and displays the results in the Analysis - Plate View area.

 

Failed syringes are indicated by red cells. A list of the failed syringes is located below the analysis results.

 

10	To verify that failures are defective syringes, go to Verifying defective syringes.

11	To save the results of the test:

a	Click Save Report.

b	In the Save As dialog box, navigate to where you want to save the results.

c	Change the file name, if desired, and click Save.

 

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Verifying defective syringes

Use the following procedure to verify that failed test results indicate defective syringes and not a non-syringe-related problem.

This procedure specifies volume requirements for the default labware selections. If you choose different labware types from the list of options, you should adjust the volumes accordingly.

Before you start

•	Verify that the wash station has a sufficient supply of source water. If necessary, refill the source bottle.

•	Prime the wash station by running the Startup protocol. See Running the Startup protocol.

•	Rotate the rack of test cartridges at location 6 by 180° so that the cartridge that was in the A1 position is now in the H12 position.

•	Add 65 mL of water to the remainder of the liquid in the Water Reservoir at deck location 4.

•	Add 22 mL of Test Solution to the remainder of the liquid in the Test Solution Reservoir at location 7.

Procedure

To verify defective syringes:

1	Rerun the Syringe Test utility protocol. See Running the syringe test.

2	Analyze the data. See Analyzing the results.

3	Compare the results to the first test:

•	If a syringe fails both tests, the syringe is defective and should be replaced. See Replacing defective syringes for repair options.

•	If a failing test result is not replicated, the syringe is acceptable for use. The initial failing result might have been the result of a non-syringe related problem.

For example, a faulty cartridge will give a failing result on both the test and retest, but the location of the failure will track with the cartridge. This is why the cartridge rack is rotated by 180-degrees before the verification test.

 

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Replacing defective syringes

To replace damaged syringes, do one of the following:

•	Use the AssayMAP Syringe Replacement Kit (part number G5409-68002), and follow the included instructions.

•	Contact Agilent Technical Support to schedule a service call.

If you continue to use the AssayMAP Bravo Platform while awaiting repair, do not use data obtained from the defective syringe.

 

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