Hello, I am doing some testing on lateral flow devices/materials and have run into a slight issue where I do not understand what the cause may be, but I have some ideas.

General Experimental details are below along with the differences.

**Day 1 (Assume Monday):**

1. I make a traditional lateral flow assay according to commercial guidelines. I have a sheet where I can get about 20 devices.

2. The lateral flow assay is made with untreated un-blocked conjugate pads (GFCP203000 from Millipore Sigma). This is for experimental purposes which is disclosed information.

3. I pipet a sample volume onto the conjugate pad (assume 50 µL for now) and it is absorbed into the pad immediately.

4. I proceed with the rest of the assay instructions.

5. I am left with 10 devices to test the next day.

**Day 2 (Assume Tuesday of the same week):**

1. I take the leftover devices from Day 1 (again, this is the same device made with untreated un-blocked conjugate pads [GFCP203000 from Millipore Sigma]).

2. I pipet the same size sample volume (50 µL) onto the conjugate pad, but it forms a bulbous drop and takes some time for the pad to absorb it (about 16-20 seconds after initial drop).

3. The entire pad does not uptake the sample (it is expected to be absorbed into the whole pad); it only gets absorbed into the half that is closest to the nitrocellulose.

4. Nothing from the original steps has changed.

I know these are the exact same devices I made as I packaged, sealed, and labeled them with my name and date of make. I did not intentionally change anything and it does not look like anyone else opened or used them.

The new conjugate pads currently in the same original Millipore Sigma package are behaving as expected (desired) like in the first experimental trial. So, it seems it is only the devices I have made. The only things I can think of that might be contributing to the new behavior are:

1. The room is slightly warmer/humid compared to the day before.

2. I slid the conjugate pad out of the package, perhaps causing some static buildup, although this did not seem to be an issue on Day 1 testing.

3. I kept the devices in a plastic protective sleeve and pulled them out, possibly introducing static that might be preventing the fluid from seeping into the pad more easily.

4. The plastic is slightly hydrophobic on the inside; if the heat and humidity in the room were high enough (although I believe it is no more than 40% humidity and 60-70°F), could some of the coating have transferred onto the exposed conjugate?

Any insight or ideas are extremely helpful!

Keywords: Lateral flow devices, Conjugate pad (GFCP203000), Millipore Sigma, Untreated, un-blocked, Sample volume, Absorption, hydrophobic, Environmental changes (temperature, humidity), Static buildup, Hydrophobic coating transfer, Assay instructions, Testing issues, Nitrocellulose membrane, Point of care devices, point of care testing.