Micro-systems for time-resolved fluorescence analysis using CMOS single-photon avalanche diodes and micro-LEDs

Abstract

Fluorescence based analysis is a fundamental research technique used in the life sciences. However, conventional fluorescence intensity measurements are prone to misinterpretation due to illumination and fluorophore concentration non-uniformities. Thus, there is a growing interest in time-resolved fluorescence detection, whereby the characteristic fluorescence decay time-constant (or lifetime) in response to an impulse excitation source is measured. The sensitivity of a sample’s lifetime properties to the micro-environment provides an extremely powerful analysis tool. However, current fluorescence lifetime analysis equipment tends to be bulky, delicate and expensive, thereby restricting its use to research laboratories. Progress in miniaturisation of biological and chemical analysis instrumentation is creating low-cost, robust and portable diagnostic tools capable of high-throughput, with reduced reagent quantities and analysis times. Such devices will enable point-of-care or in-the-field diagnostics. It was the ultimate aim of this project to produce an integrated fluorescence lifetime analysis system capable of sub-nano second precision with an instrument measuring less than 1cm3, something hitherto impossible with existing approaches. To accomplish this, advances in the development of AlInGaN micro-LEDs and high sensitivity CMOS detectors have been exploited. CMOS allows electronic circuitry to be integrated alongside the photodetectors and LED drivers to produce a highly integrated system capable of processing detector data directly without the need for additional external hardware. In this work, a 16x4 array of single-photon avalanche diodes (SPADs) integrated in a 0.35μm high-voltage CMOS technology has been implemented which incorporates two 9-bit, in-pixel time-gated counter circuits, with a resolution of 400ps and on-chip timing generation, in order to directly process fluorescence decay data. The SPAD detector can accurately capture fluorescence lifetime data for samples with concentrations down to 10nM, demonstrated using colloidal quantum dot and conventional fluorophores. The lifetimes captured using the on-chip time gated counters are shown to be equivalent to those processed using commercially available external time-correlated single-photon counting (TCSPC) hardware. A compact excitation source, capable of producing sub-nano second optical pulses, was designed using AlInGaN micro-LEDs bump-bonded to a CMOS driver backplane. A series of driver array designs are presented which are electrically contacted to an equivalent array of micro-LEDs emitting at a wavelength of 370nm. The final micro-LED driver design is capable of producing optical pulses of 300ps in width (full width half maximum, FWHM) and a maximum DC optical output power of 550μW, this is, to the best of our knowledge, the shortest reported optical pulse from a CMOS driven micro-LED device. By integrating an array of CMOS SPAD detectors and an array of CMOS driven AlInGaN micro-LEDs, a complete micro-system for time-resolved fluorescence analysis has been realised. Two different system configurations are evaluated and the ability of both topologies to accurately capture lifetime data is demonstrated. By making use of standard CMOS foundry technologies, this work opens up the possibility of a low-cost, portable chemical/bio-diagnostic device. These first-generation prototypes described herein demonstrate the first time-resolved fluorescence lifetime analysis using an integrated micro-system approach. A number of possible design improvements have been identified which could significantly enhance future device performance resulting in increased detector and micro-LED array density, improved time-gate resolution, shorter excitation pulse widths with increased optical output power and improved excitation light filtering. The integration of sample handling elements has also been proposed, allowing the sample of interest to be accurately manipulated within the micro-environment during investigation.