Traditional biochemical methods for enzyme detection are mainly based on antibody-based immunoassays, which lack the ability to monitor the spatiotemporal distribution and, in particular, the in situ activity of enzymes in live cells and in vivo. In this review, we comprehensively summarize recent progress that has been made in the development of small-molecule as well as material-based fluorogenic probes for sensitive detection of the activities of enzymes that are related to a number of human diseases. The principles utilized to design these probes as well as their applications are reviewed. Specific attention is given to fluorogenic probes that have been developed for analysis of the activities of enzymes including oxidases and reductases, those that act on biomacromolecules including DNAs, proteins/peptides/amino acids, carbohydrates and lipids, and those that are responsible for translational modifications. We envision that this review will serve as an ideal reference for practitioners as well as beginners in relevant research fields.

Fluorogenic probes for disease-relevant enzymes.

The diagnosis of viral infections by culture relies on the collection of proper specimens, proper care to protect the virus in the specimens from environmental damage, and use of an adequate transport system to maintain virus activity. Collection of specimens with swabs that are toxic to either virus or cell culture should be avoided. A variety of transport media have been formulated, beginning with early bacteriological transport media. Certain swab-tube combinations have proven to be both effective and convenient. Of the liquid transport media, sucrose-based and broth-based media appear to be the most widely accepted and used. Studies on virus stability show that most viruses tested are sufficiently stable in transport media to withstand a transport time of 1 to 3 days. Some viruses may withstand longer transport times. In many cases, it is not necessary to store virus specimens in a refrigerator or send them to the laboratory on wet ice or frozen on dry ice. However, the specimen should not be exposed to environmental extremes. Modern viral transport media allow for more effective use of viral culture and culture enhancement techniques for the diagnosis of human viral infections.

Transport of viral specimens.

Recombinant adeno-associated viral (AAV) capsid proteins are provided. Methods for generating the recombinant adeno-associated viral capsid proteins and a library from which the capsids are selected are also provided.

Aav capsid library and aav capsid proteins

A new endonuclease/methyltransferase activity assay method based on graphene oxide (GO) is developed. Substrate DNA is designed to possess a double-stranded part to serve as a nuclease substrate and a single-stranded part for anchoring the DNA to the GO surface via strong noncovalent binding. Nuclease-mediated DNA hydrolysis induces the recovery of fluorescence intensity of the dye attached to the end of the double-stranded DNA region. This GO-based method allows real-time measurement and quantitative assay for endonuclease/methyltransferase activities in short time.

A New Assay for Endonuclease/Methyltransferase Activities Based on Graphene Oxide

A new label-free strategy for sensitive fluorometric biosensing based on perylene probe monomer–excimer transition has been developed. A negatively charged perylene probe (compound 1) was used. Compound 1 shows strong monomer fluorescence in an aqueous buffer solution. A cationic polymer could induce aggregation of compound 1 through noncovalent interactions. Compound 1 monomer emission was quenched, and meanwhile strong excimer emission was observed. Upon addition of a single-stranded DNA (an anionic polymer), strong electrostatic attractive interactions between the cationic polymer and the DNA weakened the binding of aggregates of compound 1 to the polycation. Compound 1 monomers were released, and excimer–monomer emission transition was detected. This observation formed the basis for DNA methyltransferase (MTase) activity detection. When the 3′-OH terminus of a duplex DNA was removed, the DNA strands could not be elongated by terminal deoxynucleotidyl transferase (TdT), and little restoration of compou...

Polymer-induced perylene probe excimer formation and selective sensing of DNA methyltransferase activity through the monomer-excimer transition.

An enzyme linked immunosorbent assay (ELISA) system which detects and simultaneously types herpes simplex virus antigens in clinical specimens from patients with genital herpes has been compared with standard tissue culture isolation. Although more sensitive than a similar method previously described and also more sensitive than electron microscopy and immunofluorescence, ELISA did not detect all the viruses isolated in tissue culture. Costs were comparable. The speed of obtaining the result together with knowledge of the type causing infection are useful when antiviral chemotherapy is envisaged and when considering the likelihood of recurrences.

https://jcp.bmj.com/content/jclinpath/38/5/554.full.pdf

Comparison of ELISA with virus isolation for the diagnosis of genital herpes.

The continuous cell line 293 was evaluated as a replacement for primary human embryo kidney (HEK) cells in the cultivation of ocular viruses. The 293 cells were found to be as sensitive as HEK cells and human embryo fibroblast (HEF) cells for the cultivation of adenoviruses and herpes simplex virus (HSV) respectively. As a continuous cell line, 293 cells are preferable to HEK and HEF cells for the routine isolation of ophthalmic viruses.

https://jcp.bmj.com/content/jclinpath/36/9/996.full.pdf

Comparison of the continuous cell line 293 with human embryo kidney cells and human embryo fibroblast cells for the cultivation of ocular viruses.

Isolates of herpes simplex virus which had previously been typed by restriction enzyme analysis were typed again with a commercial ELISA system using polyclonal antibodies. There was complete correlation between the two techniques. Although restriction is more precise and definitive, when typing only is required the simplicity of ELISA makes it the preferred technique.

https://jcp.bmj.com/content/jclinpath/37/8/937.full.pdf

J. Harbour

Papers

Comparison of ELISA with virus isolation for the diagnosis of genital herpes.

Comparison of the continuous cell line 293 with human embryo kidney cells and human embryo fibroblast cells for the cultivation of ocular viruses.

Comparison of a commercial ELISA system with restriction endonuclease analysis for typing herpes simplex virus.

Comparison ofthecontinuous cell line 293with humanembryokidney cells andhumanembryo fibroblast cells forthecultivation ofocular viruses