Parallel Fluorescence Detection Research Articles

Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection

We report a 3D microfluidic device with 32 detection channels and 64 sheath flow channels and embedded microball lens array for high throughput multicolor fluorescence detection. A throughput of 358 400 cells/s has been accomplished. This device is realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. High refractive index micro ball lenses (n = 2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets.

Highly Sensitive and Selective Bifunctional Oligonucleotide Probe for Homogeneous Parallel Fluorescence Detection of Protein and Nucleotide Sequence

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.

Simultaneous near-field and far-field fluorescence microscopy of single molecules

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

Monolithic silicon matrix detector with 50 μm photon counting pixels

A 48-element monolithic matrix detector, designed for parallel fluorescence detection, has been fabricated in silicon and tested. The pixels are single photon avalanche diodes (SPADs) with 50 µm diameter, ordered in a 6× 8 array with 240 µm pitch. The photon detection efficiency is remarkably uniform over the array: it has a peak of 48% at 530 nm and it is higher than 30% over all the visible range. Low dark counting rate (DCR) is obtained in operation at moderately low temperature (−15°C with thermoelectric cooling): the individual pixel DCR is 60 c s−1 for about 40% of the elements and it is below 5700 c s−1 in the rest of the array. It was verified that the afterpulsing probability is below 1% and that the optical crosstalk probability between elements is lower than 0.2%. Based on this matrix detector, we have developed a versatile and compact (20 cm× 8 cm × 4 cm) photon counting module that can be easily interfaced to a PC via USB link.

Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2 x 2 fan-out element.

We have developed a multifocal diffractive-optical fluorescence correlation spectroscopy system for parallel excitation and detection of single tetramethylrhodamine biomolecules in microarrays. Multifocal excitation was made possible through the use of a 2 x 2 fan-out diffractive-optical element with uniform intensity in all foci. Characterization of the 2 x 2 fan-out diffractive-optical element shows formation of almost perfect Gaussian foci of submicrometer lateral diameter, as analyzed by thermal motion of tetramethylrhodamine dye molecules in solution. Results of parallel excitation and detection in a high-density microarray of circular wells show single-biomolecule sensitivity in all four foci simultaneously.

Cross-talk reduction in confocal images of dual fluorescence labelled cell spheroids

Dual fluorescence labelling is an advanced method to separate two individual specimens in a biological system using confocal microscopy. An inherent problem of this method is fluorescence channel cross-talk, which causes problems for the exact spatial determination and separation of the specimens. Using a parallel fluorescence detection and an image processing technique, based on an image subtraction method, we have developed a very straight forward method for correcting the dual channel fluorescence images. We successfully applied this method to a 3-dimensional cancer spheroid invasion assay and controlled the cross-talk compensation efficiency by a quality parameter.

Detection System for Reaction-Rate Analysis in a Low-Volume Proteinase-Inhibition Assay

High-throughput screening of large combinatorial chemical libraries in biochemical assays will benefit from reduced reagent volume and increased speed of measurement. Standard assays typically are performed in 96-well microtiter plates having 200-μL well volumes and up to an hour of incubation time. In this paper, we demonstrate a technique for precise and rapid measurement of the progress of an enzymatic reaction and its inhibition with reduced volume and time (for this work, the assay was mixed at the 200-μL level and detected in 2-μL volumes with minutes of total assay time). Directly measuring the enzyme activity in the small volume format yields a precise value for the median inhibitory concentration (IC50) of an inhibitor compound. The model assay is the endoproteolytic cleavage of a small fluorogenic peptide by human neutrophil collagenase (MMP-8). The fluorogenic peptide was labeled at one end with a UV/blue fluorophore (N-methylanthranilyl) and at the other end with a quencher (dinitrophenol). To generate inhibition data, a hydroxamate peptide analog inhibitor of collagenase, actinonin, was included in the reaction. The experiments were performed using ultraviolet laser illumination (325 nm wavelength) and parallel fluorescence detection by a cooled, charge-coupled-device camera system to increase sensitivity and speed. The assay volume was reduced to 2 μL for data collection, and the total time for mixing, incubation, and measurement was less than 6 min. For comparison to a standard format, the same assay was performed in a 96-well microtiter plate in 200 μL using 30 min of incubation and measurement in a microtiter plate fluorimeter. Median inhibitory concentrations (IC50) for actinonin of 73 ± 16 and 100 ± 14 nM were obtained in the 2- and 200-μL assays, respectively. One concern with assay miniaturization and increases in throughput is a potential loss of precision and accuracy. Laser excitation and parallel detection of fluorescence is a promising approach for increased speed and reduced cost without loss of precision for proteinase inhibition assays.