Ultrasensitive detection of proteins and sugars at single-cell level

Abstract

Each cell produces its own responses even if it appears identical to other cells. To analyze these individual cell characteristics, we need to measure trace amounts of molecules in a single cell. Nucleic acids in a single cell can be easily amplified by polymerase chain reaction, but single-cell measurement of proteins and sugars will require de novo techniques. In the present study, we outline the techniques we have developed toward this end. For proteins, our ultrasensitive enzyme-linked immunosorbent assay (ELISA) coupled with thionicotinamide-adenine dinucleotide cycling can detect proteins at subattomoles per assay. For sugars, fluorescence correlation spectroscopy coupled with glucose oxidase-catalyzed reaction allows us to measure glucose at tens of nM. Our methods thus offer versatile techniques for single-cell-level analyses, and they are hoped to strongly promote single-cell biology as well as to develop noninvasive tests in clinical medicine.

Keywords: HIV-1 p24; adiponectin; fluorescence correlation spectroscopy; insulin; thio-NAD cycling.

Figure 1.

Scheme of ultrasensitive detection of proteins by ELISA coupled with thio-NAD cycling. Our ultrasensitive ELISA requires only the addition of a thio-NAD cycling solution, which includes androsterone derivatives, and 3α-hydroxysteroid dehydrogenase and its coenzymes, to the usual ELISA without any use of special instruments. Absorption (400 nm) of thio-NADH is measured with a commercially available microplate reader.

Figure 2.

Scheme of ultrasensitive detection of sugars by FCS.The concentrations of H₂O₂ correspond to those of the proteins labeled with tyramide-tetramethyl rhodamine (TMR). H₂O₂ is produced by the reaction between glucose and glucose oxidase, and thus we can determine the concentrations of H₂O₂ and deduce the concentrations of glucose by FCS.