Shine bright: considerations on the use of fluorescent substrates in living monoaminergic neurons in vitro

The biogenic monoamines dopamine （DA）, norepinephrine （NE） and serotonin （5-hydroxytryptamine, 5-HT） are major neuromodulators in the mammalian central nervous system （CNS）. DA containing neurons are found in i） the mesolimbic system in which cell bodies in the ventral tegmental area （VTA） project axons into the amygdala, cortex, hippocampus and the nucleus accumbens; and ii） the nigrostriatal system in which cell bodies located in the substantia nigra pars compacta send their axons into the dorsolateral parts of the striatum （Bjorklund and Dunnett, 2007）. The central noradrenergic neurons are concentrated in distinct brainstem nuclei with the locus coeruleus （LC） being the most prominent nucleus which projects a diffusely arborizing axonal network to most areas of the CNS （Szabadi, 2013）.

Tetra-substituted Amino Aluminum Phthalocyanine as a New Red-region Fluorescent Substrate for Horseradish Peroxidase Based Enzyme-linked Immunosorbent Assay

The use of tetra-substituted ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis-Menten parameters Km and Vmax were measured to be 2.82 × 10?6 mol/L?1 and 6.0 × 10?9 mol·L?1, respectively. In this paper, TAAIPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5–200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.

Developing a fluorescence substrate for HRP-based diagnostic assays with superiorities over the commercial ADHP

The combination of horseradish peroxidase(HRP)and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays.10-Acetyl-3,7-dihydroxyphenoxazine(ADHP or Amplex red)is the most sensitive fluorogenic substrate known for HRP in current market,however,it suffers from some drawbacks,such as non-specific reactivity to carboxylesterase and limited fluorescence stability.In the present study,a novel HRP substrate10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine(AR-2),has been prepared,which exhibited improved sensitivity than ADHP in sensing HRP.Moreover,the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant p H fluctuation as compared to ADHP/HRP.Successful detection of uric acid/urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring H_(2)O_(2)-generating biochemical reactions.More interestingly,an enzyme-linked immunosorbent assay(ELISA)using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)from human serum samples.Overall,AR-2 exhibits improved performances over the commercial ADHP,which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.