High-Resolution Near-Infrared Imaging of DNA Microarrays with Time-Resolved Acquisition of Fluorescence Lifetimes

Abstract

Ultrasensitive, near-infrared (NIR), time-resolved fluorescence is evaluated as a detection method for reading DNA hybridization events on solid surfaces for microarray applications. In addition, the potential of mulitiplexed analyses using time-resolved identification protocols is described. To carry out this work, a NIR time-resolved confocal imager was constructed to read fluorescence signatures from the arrays. The device utilized a 780-nm pulsed diode laser, a single-photon avalanche diode (SPAD), and a high-numerical-aperture microscope objective mounted in an epi-illumination format. Due to the small size of the components that are required to construct this imager, the entire detector could easily be mounted on high-resolution translational stages and scanned over the stationary arrays. The instrument response function of the device was determined to be 275 ps (fwhm), which is adequate for measuring fluorophores with subnanosecond lifetimes. To characterize the system, NIR dyes were deposited directly on different substrate materials typically used for DNA microarrays, and the fluorescence lifetimes of two representative dyes were measured. The fluorescence lifetime for aluminum tetrasulfonated naphthalocyanine was found to be 1.92 ns, and a value of 1.21 ns was determined for the tricarbocyanine dye, IRD800, when it was deposited onto poly(methyl methacrylate) (PMMA) and measured in the dry state. Finally, the imager was used to monitor hybridization events using probe oligonucleotides chemically tethered to a PMMA substrate via a glutardialdehyde linkage to an aminated-PMMA surface. The limit of detection for oligonucleotides containing a NIR fluorescent reporter was determined to be 0.38 molecules/microm2, with this detection limit improving by a factor of 10 when a time-gate was implemented. Fluorescence lifetime analysis of the hybridization events on PMMA indicated a lifetime value of 1.23 ns for the NIR-labeled oligonucleotides when using maximum-likelihood estimators.

A Substrate‐Independent Approach for the Surface Immobilization of Oligonucleotides using Aldehyde Functionalized Surfaces

“…[42] As well as its wide use in classic synthetic chemistry, Schiff-base chemistry has also proven to be popular for the immobilization of biomolecules such as DNA [12,17] or proteins [43] to aldehyde-functionalized surfaces. Mechanistically, the Full Paper amine group reacts with a carbonyl centre followed by the elimination of water to yield an imine intermediate.…”

“…[6] Typically, the oligonucleotides are assembled onto a solid surface either by in situ synthesis [7,8] or robotic spotting of whole strands. [6] Surface functionality plays a key role during microarray fabrication [9] and has always, in the past, been limited to specific substrate materials and geometries, e.g., selfassembled monolayers (SAMs) on glass, [10][11][12] silicon, [13] or gold; [14][15][16] Schiff-base derivatization of aminated PMMA polymer; [17] or alcoholysis of surface-generated acyl halides on diamond. [18] In this article we describe a substrate-independent methodology for covalent immobilization of singlestrand oligonucleotides onto solid surfaces, Scheme 1.…”

Applications of a single-molecule detection in early disease diagnosis and enzymatic reaction study

“…Early single-molecule research mainly used rhodamine- [36][37][38][39][40][41] and cyanine-derivatives. [42][43][44][45] Fluorescein isothiocyanate (FITC), a derivative of fluorescein, became popular since the excitation wavelength matches well with current lasers. [46][47][48][49] The new generation of fluorophores is generally more photostable, brighter, and less pH-sensitive than common dyes of comparable excitation and emission spectra.…”

Fluorescence Lifetime Imaging Microscope Consisting of a Compact Picosecond Dye Laser and a Gated Charge-Coupled Device Camera for Applications to Living Cells

“…In the first approach, the fluorescence lifetime is measured by means of time-correlated single-photon counting (TCSPC), and the laser beam is scanned for measuring the cell image. [1][2][3][4][5][6][7] A fluorescence lifetime image can be obtained with a temporal resolution of several tens of picoseconds, when a highly-repetitive laser having a short pulse width is used. Unfortunately, the time required to measure the lifetime image is relatively long, e.g., 30 min, and is dependent on the area of the image.…”