In general, I think last author. I'm pretty close to your field, though at a similar career stage, so I'm not the person making the decisions on your grants, so take this with that in mind! I'm also going to answer for the US, since that's the culture I know best.
In my experience, biophysics follows the cond-mat tradition of last author = group leader. Therefore, you being last author with a PD/grad student first author could indicate that this is a project that you led, and a student that you mentored, and that your PD advisor acknowledges this as "your" project. If there are only two authors, I think it doesn't matter as much. Being last could mean that you were the project lead, or that you left the manuscript in such a bad state that your PD advisor had to do most of the editing and then put him/herself as first!
I think the strongest sign of independence would be: your trainee first and you last. The worst sign would be you first, your PD advisor last (i.e. you are still just following their instructions, rather than developing your own topics.)
One caveat: that analysis assumes you're at an R1 or otherwise have students/PDs who can contribute first-author-level effort. I've also seen: trainee moves to a position at a non-R1, and doesn't have graduate students, but continues a collaboration, and usually publishes papers with former PD first, then UG trainees, and PD advisor last. I don't know how common that is, though, and if you're in that situation, it might be worth mentioning to your department chair that that's how it works, and that you are not just salami-slicing your PD work.