5(6)-Carboxyfluorescein (5(6)-FAM)

Labeling a protein with fluorophores using NHS ester derivitization

N-hydroxysuccinimde (NHS) ester-mediated derivitization involves the reaction of this amine-reactive group with the primary amines of a protein or a biomolecule. Using NHS chemistry allows one to conjugate various fluorescent probes, biotin, and cross-linkers to primary amines. For example, we use NHS ester chemistry to fluorescently label the amino terminus of a protein with the dye, 5-(and-6)-carboxyfluorescein, succinimidyl ester (5(6)-FAM, SE).

Double-Resonant Nanostructured Gold Surface for Multiplexed Detection

A novel double-resonant plasmonic substrate for fluorescence amplification in a chip-based apta-immunoassay is herein reported. The amplification mechanism relies on plasmon-enhanced fluorescence (PEF) effect. The substrate consists of an assembly of plasmon-coupled and plasmon-uncoupled gold nanoparticles (AuNPs) immobilized onto a glass slide. Plasmon-coupled AuNPs are hexagonally arranged along branch patterns whose resonance lies in the red band (?675 nm). Plasmon-uncoupled AuNPs are sprinkled onto the substrate, and they exhibit a narrow resonance at 524 nm. Numerical simulations of the plasmonic response of the substrate through the finite-difference time-domain (FDTD) method reveal the presence of electromagnetic hot spots mainly confined in the interparticle junctions. In order to realize a PEF-based device for potential multiplexing applications, the plasmon resonances are coupled with the emission peak of 5-carboxyfluorescein (5-FAM) fluorophore and with the excitation/emission peaks of cyanine 5 (Cy5). The substrate is implemented in a malaria apta-immunoassay to detect Plasmodium falciparum lactate dehydrogenase (PfLDH) in human whole blood. Antibodies against Plasmodium biomarkers constitute the capture layer, whereas fluorescently labeled aptamers recognizing PfLDH are adopted as the top layer. The fluorescence emitted by 5-FAM and Cy5 fluorophores are linearly correlated (logarithm scale) to the PfLDH concentration over five decades. The limits of detection are 50 pM (1.6 ng/mL) with the 5-FAM probe and 260 fM (8.6 pg./mL) with the Cy5 probe. No sample preconcentration and complex pretreatments are required. Average fluorescence amplifications of 160 and 4500 are measured in the 5-FAM and Cy5 channel, respectively. These results are reasonably consistent with those worked out by FDTD simulations. The implementation of the proposed approach in multiwell-plate-based bioassays would lead to either signal redundancy (two dyes for a single analyte) or to a simultaneous detection of two analytes by different dyes, the latter being a key step toward high-throughput analysis.

Fluorescent Properties of Carboxyfluorescein Bifluorophores

Bright fluorescent probes with enhanced intensities in the fluorescein channel are of great value for plenty of biological applications. To design effective probes one should introduce as many as possible fluorophores to the biomolecule while leaving its native structure as intact as possible. To reach this compromise, we designed and synthesized fluorescein bifluorophores on the 3,5-diaminobenzoic acid scaffold, which allows for insertion of two fluorophores at one modification site of a biomolecule. Rigid structure of the branching linker group allows to minimize self-quenching the fluorophores. However, despite the structure similarities of fluorescein isomers (5-FAM and 6-FAM), different photophysical behavior was observed for the corresponding bifluorophores. Here we made efforts to get insight into these effects with the focus on the media viscosity impact.

Organic Anion Detection with Functionalized SERS Substrates via Coupled Electrokinetic Preconcentration, Analyte Capture, and Charge Transfer

Detecting ultralow concentrations of anionic analytes in solution by surface-enhanced Raman spectroscopy (SERS) remains challenging due to their low affinity for SERS substrates. Two strategies were examined to enable in situ, liquid phase detection using 5(6)-carboxyfluorescein (5(6)-FAM) as a model analyte: functionalization of a gold nanopillar substrate with cationic cysteamine self-assembled monolayer (CA-SAM) and electrokinetic preconcentration (EP-SERS) with potentials ranging from 0 to +500 mV. The CA-SAM did not enable detection without an applied field, likely due to insufficient accumulation of 5(6)-FAM on the substrate surface limited by passive diffusion. 5(6)-FAM could only be reliably detected with an applied electric field with the charged molecules driven by electroconvection to the substrate surface and the SERS intensity following the Langmuir adsorption model. The obtained limits of detection (LODs) with an applied field were 97.5 and 6.4 nM on bare and CA-SAM substrates, respectively. For the CA-SAM substrates, both the ligand and analyte displayed an ?15-fold signal enhancement with an applied field, revealing an additional enhancement due to charge-transfer resonance taking place between the metal and 5(6)-FAM that improved the LOD by an order of magnitude.

Ratiometric Fluorescent Metal-Organic Framework Biosensor for Ultrasensitive Detection of Acrylamide

Acrylamide is a neurotoxin and carcinogen that forms during the thermal processing of food, inflicting irreversible harm to human health. Herein, a ratiometric fluorescence biosensor based on a 6-carboxyfluorescein-labeled aptamer (FAM-ssDNA) and porphyrin metal-organic framework (PCN-224) was developed. PCN-224 exhibits strong adsorption capacity for FAM-ssDNA and also quenches the fluorescence of FAM-ssDNA via fluorescence resonance energy transfer and photoinduced electron transfer. FAM-ssDNA hybridizes with complementary DNA to form double-stranded DNA (FAM-dsDNA), which is liberated from the PCN-224 surface, resulting in fluorescence recovery. However, the intrinsic fluorescence of the ligand remains unchanged. Acrylamide can create an adduct with FAM-ssDNA and inhibit the hybridization of FAM-dsDNA, thus realizing ratiometric sensing of acrylamide. The proposed biosensor displays excellent detection performance from 10 nM?0.5 mM with a limit of detection of 1.9 nM. In conclusion, a fabricated biosensor was successfully applied to detect acrylamide in thermally processed food, and the results were consistent with those of high-performance liquid chromatography.