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This paper presents a novel auto-calibration technique for eliminating sensor mismatch in CMOS-based chemical imagers. Designed using an 8 × 8 array comprising of pH-sensitive ion-sensitive field-effect transistors (ISFETs), the chemical imager is capable of implementing a gradient-based calibration algorithm by biasing programmable-gate (PG) ISFETs at a common operating point when exposed to a solution of homogenous pH. The system was fabricated in a typical 0.35-μm CMOS technology and demonstrated a fast rate of convergence (500 ms per iteration) while a convergence accuracy of 45 mV on a gain of 10 (0.5% relative standard error and 2% pixel-to-pixel variation) was achieved. A maximum pH sensitivity of 57 mV/pH is also reported.