To quote the wiki:
To quote the Professors Foglio, the Spark is "whatever it is that makes Mad Scientists what they are. A poorly understood concept that identifies and incorporates a batch of personality traits shared by those who have it." They are "people who seem to have the ability to tinker with the laws of physics as we know them. They are brilliant, focused, and often impatient with those whose thoughts don't run with the speed or in the direction of their own. Because of this, some of those thoughts have veered off in truly alarming directions. This makes them dangerous and shortsighted..."
There can be competent, even brilliant, engineers and researchers who are nevertheless not Sparks; and therefore will never make the intuitive leaps necessary to "truly warp the laws of nature" and arrive at such creations as Dr. Jekyll's formula, Dr. Frankenstein's monster, a Strong AI implemented in clockwork, or even the absolutely perfect cup of coffee. (However, some apparently sparky creations — such as nuclear submarines equivalent to Captain Nemo's Nautilus, heavier-than-air flying machines, Google AdSense, and mortgage-backed securities — actually do exist in our world.)
There is no distinct physical sign (other than a surprising number of Sparks have chosen to alter themselves, perhaps as repair from failed experiments). The trait does seem to run in families, so it may be genetic. Baron Wulfenbach believes there's a part of the brain that governs it that can be excised, but he hasn't had a lot of luck. Recently, a seeming success in lobotomization, Dimitri Vapnoodle, has seemingly revealed that he still has the Spark (although the circumstances were such that he might have been healed by an outside force). This could also be tied to the general greater physical hardiness of Sparks, a matter of his brain knitting itself back together again. At this time, the situation is unanswered.