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A Time-Gated Fluorescence Instrument and Its Applications Using Mn-Doped I(II)-III-VI Nanocrystal Probes
Electrical and Biomedical Engineering
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In this work, two versions of a low-cost, portable, time-gated fluorescence intensity measurement instrument were developed with the primary aim of utilizing Mn-doped I(II)-III-VI nanocrystals (Mn:AgZnInS/ZnS and Mn:CuZnInS/ZnS core/shell) as long-lifetime fluorescent probes for sensing applications. These nanocrystals have several advantages over other commonly used fluorophores, including stability in a wide range of solvent conditions and capacity for bioconjugation with established surface modification procedures, excitation with inexpensive visible-light sources, high brightness, minimal reabsorption, and fluorescence lifetimes up to milliseconds. These properties allow for a wide range of applications, as well as simple and inexpensive instrument design. The first version of the developed instrument was designed for large-volume samples within glass cuvettes. The time-gated measurement method was implemented using a central microcontroller unit for laser pulse and detector timing control. The instrument was found to have a large dynamic range of nearly six orders of magnitude with detector sensitivity adjustments and to be highly sensitive even in high autofluorescence sample matrices, with limit of detection (LOD) ~40 pg/mL for Mn:AgZnInS/ZnS nanocrystals in an aqueous 5% bovine serum albumin solution. In a fluorescence quenching detection assay for copper (II) ion detection in rum – a high-autofluorescence, dark-colored, complex sample matrix for which many conventional detection methods are inappropriate – the observed LOD of copper ions (~2 ppb) was found to be much lower than those allowed in beverages within the U.S. (1.3 ppm), indicating potential applicability of this method to general food and drink testing. The second version of the instrument was developed for small-volume or surface-based measurements for applications such as high-throughput plate-based assays or disposable paper strip-based lateral flow immunoassays. The Mn-doped I(II)-III-VI fluorescent probes, despite their similar physical properties, were found to have significantly different LODs from their raw fluorescence signal calibrations (AgZnInS 354 pg/mL and CuZnInS 2.9 ng/mL) and a simple microwell surface-based biotin-avidin binding assay (316 ng/mL and 1025 ng/mL). Based on these results, Mn:AgZnInS/ZnS nanocrystals alone were utilized in a magnetic bead-based sandwich immunoassay for the Burkholderia pseudomallei capsular polysaccharide antigen using Pf4c4 monoclonal antibody. The assay was found to be very sensitive (2σ LOD at 42.54 pg/mL, N = 9). Notably, this assay did not involve the use of enzyme-based methods, which tend to add significant cost and may suffer stability issues even in mild storage conditions.