There is no fixed minimum. The amount is weighed against the total cost of buying or establishing this business; buying an existing clinic outright usually meets it. The remaining question is whether the money is committed.
Substantial is relative to this business
Hypothetical example: a Quispamsis buyer is purchasing an existing clinic for a price that reflects its equipment, lease and patient base, with no additional build-out needed. When the investor pays substantially the whole cost of acquiring an established business, the investment is usually substantial in proportion to the enterprise's cost.
The more useful cost questions are practical: the consular application fee for the E-2 visa is set by the State Department; the franchise transfer fee, legal review of the franchise agreement, escrow fees and the cost of state licensure for the investor are business costs that should appear in the plan. If part of the price is deferred or seller-financed, only the amounts actually committed and at risk count, so the structure of the deal affects the immigration analysis as well as the price.