Enzyme Substrates for Diagnostics

Microbiology research, clinical diagnostics, food manufacturing and environmental testing all call for a reliable and efficient way to detect and identify bacterial species. This has become an even bigger necessity in recent years, due to the healthcare industry being threated by multidrug-resistant bacterial pathogens. Currently diagnostic methods can use the bacteria's nucleic acid sequences, antigens or enzymes to identify them. One commonly used method, identifying bacteria through specific nucleic acid sequences, is the PCR-based molecular method. However this method is limited in that it cannot determine if the bacteria is dead or viable and whether the protein of the gene it has detected is actually expressed. It also requires the design of new primers for the target gene, which is not possible for new clinical isolates with genetic mutations. One possible solution to overcome these disadvantages, is to use enzyme substrates to identify the enzymes in bacteria.   

Over the years, Biosynth has developed a range of enzyme substrates for applications in culture media, diagnostics, food and environmental testing. We offer a range of chromogenic, fluorogenic and bioluminescent substrates, as well as products for diagnostic probing and staining. We even have enzyme substrates, of high biochemical purity for lysosomal storage disorders. Our scientists have great expertise in synthetic chemistry and microbiology. As such, we can chemically tailor diagnostic reagents to certain types of assays and applications, so that customers are no longer held back by the limits set by the commercial choice of biochemical reagents. We are continuously building our experience and using our intellectual property by formulating licensing agreements and/or joint projects. Find below a handful of the enzyme substrates for diagnostics we offer.

Contact us today to inquire.

Aldol® indicator platform

Our Aldol® indicator platform is made up of chromogenic substrates for diagnostics. These products are largely soluble colorless molecules made up of a chromophore and a specific enzyme substrate. In the presence of a specific enzyme the chromophore-substrate conjugate is cleaved, producing an insoluble product of distinctive color. The fact that an insoluble signalophor is produced means that this detection system is well suited for use in solid bacteriological culture media as it will not diffuse through the medium. This means that a single assay can be used to detect and localize specific enzyme activity and hence individual colonies. Interestingly, more than one enzyme on a single plate can be detected through the use of Aldol® substrates. For instance when Aldol® 495 β-D-galactopyranoside is used with a 5-bromo-4-chloro-3-indoxyl-β-D-glucuronic acid substrate, in the presence of β-glucuronidase, a green-blue color is produced. However a violet color is produced from both dyes in the presence of the enzymes β-galactosidase and a β-glucuronidase enzymes, allowing for bacteria such as Klebsiella pneumoniae, E. coli and Salmonella enteritidis to be identified.

Current substrates that are in use have disadvantages in that they produce toxic intermediates and need oxygen, other oxidizers or toxic diazonium slats to be present. Biosynth's Aldol® indicator platform overcomes these challenges and can provide a range in colors from yellow to red which can easily be distinguished from the background and conventional chromogenic substrates. We even have Aldol® fluorophore dyes which come in green yellow or red. In comparison to indoxyl-, 4-MU- and fluorescein conjugates, Aldol® substrates such as Aldol® 458 have a greater ester stability and therefore have less unspecific hydrolysis. 

An example of an Aldol substrate that is both chromogenic and fluorogenic, is Aldol® 515 phosphate (A-4721_P00), which is cleaved in the presence of phosphatases enzymes, producing an intense red color. A bright fluorescence signal is obtained in the presence of 0.3 mM Aldol® 355 fluorescence enhancer (EA175365). In liquid media, the addition of 1% w/v Tween® 80 also enhances the fluorescence. 

The use of fluorescent signal in diagnostics is useful as the fluorescence signals can be separated from the sample solution after the diagnostic test. The information remains stable in the polymer for further analysis, even after the sample has been disposed of.

Find out more information on our Aldol substrates webpage.

AquaSpark® Chemiluminescence

The Biosynth AquaSpark® family of products (AquaSpark® is a trademark of Ramot at Tel Aviv University), are a collection of chemiluminescence probes and enzyme substrates for research and diagnostic applications. These products work as single agents, have higher efficiency and sensitivity over existing products and can be used ex vivo and in vivo. The AquaSpark luminophor produces the strongest light you have ever experienced in an bio-assay. This technology has excellent applications in the detection of biomarkers, metabolites, enzyme activity, reactive oxygen species or pathogens.

AquaSpark® Alkaline Phosphatase

Quantifying the levels of the Alkaline Phosphatase enzyme in the blood, can contribute to the diagnosis of some liver and bone diseases. For alkaline phosphatase activity blood serum tests (of ALP-related diseases), Biosynth offer AquaSpark® Alkaline Phosphatase Substrate (EA175391, A-8164_P00) and AquaSpark® Broad Range Phosphatase Substrates. We also have ready to use alkaline phosphatase products (EA175401, A-8177_P00) suitable for use in immunoassays such as ELISA, Western Blot and Dot Blot. 

AquaSpark® beta-D-galactoside

The enzyme beta-galactosidase catalyzes the hydrolysis of beta-d-galactoside in the presence of water, to galactose and alcohol. It is found in many organisms, plants and animals including E.coli. Although most E.coli, are harmless, some have the potential to cause illnesses such as respiratory illness, diarrhea and urinary tract infections. It is therefore important to identify the presence of harmful E.coli and other bacteria in patients, food manufacturing settings and in water supplies.

Beta-Galactosidase can be used in ELISA kits when conjugated to antibodies or streptavidin, to determine lactose in biological fluids and it is employed in food processing operations. Biosynth's AquaSpark® beta-D-galactoside (EA182509, A-8169_P00), provides a substrate for beta-Gal detection and with greater sensitivity compared to traditional methods such as the used of X-Gal or ONPG chromogenic substrates. 

AquaSpark® Peroxide Probe

Our AquaSpark® Peroxide Probe (EA181784, A-8170_P00) detects, using chemiluminescence, any H2O2-releasing oxidase activity such as monoamine oxidase, uiricase, L-amino acid oxidase and nucleoside oxidase. The detection of increased H2O2 production in cells and tissues, may help in the diagnosis of inflammation, cardiovascular, cancer and neurodegenerative diseases. The AquaSpark® Peroxide Probe when coupled to the H2O2 producing oxidase, glucose oxidase, can quantify levels of glucose in body fluids, fermentation media, food or vegetal raw material.  

AquaSpark® 510 Nitroreductase/NADH Probe

The AquaSpark® 510 Nitroreductase/NADH Probe (CL-NTR) (EA175403; A-8185_P00) is a probe for the selective chemiluminescent detection of nitroreductase activity, in bacteria or animal models. It can also be used to quantify NADH concentration in vitro when used with a defined nitroreductase enzyme concentration.

AquaSpark® alpha-D-glucoside

AquaSpark® alpha-D-glucoside (EA182766, A-8171_P00), can be used for identification of Cronobacter sakazaki, Staphylococcus aureus (including MRSA), Enterobacter spp., Bacillus stearothermophilus and further microorganisms.

AquaSpark® beta-D-glucuronide

AquaSpark® beta-D-glucuronide (EA175400, A-8175_P00) is a luminogenic substrate for highly sensitive detection of Escherichia coli, GUS reporter gene assays and further analytical methods based on the detection of beta-glucuronidase. 

AquaSpark® H2S and H2S-SH

AquaSpark® H2S (EA179527, A-8211_P00) and H2S-SH (EA179526, A-8212_P00) are chemiluminescent probes for the detection of sulphides and thiols.

Other Products Available at Biosynth

X-Shining™ Luciferase - For ATP tests in hygiene monitoring, drug screening and bacteria identification in microbial assays, Biosynth can offer our X-Shining™ Luciferase enzyme which has been optimized by genetic engineering for strongly increased thermostability and storage stability. We have our X-Shining™ all-in-one ATP Assay for the specific bioluminescent detection of ATP levels in a single vial and our X-ShiningTM AST Range which provides a rapid readout for antibiotic susceptibility. 

STAADIUM™ Inhibitors - When added to solid growth media our STAADIUM™ inhibitors inhibit the growth of unwanted bacterial background flora, allowing for easier detection of colonies of interest.

RUG® - resorufin-β-D-glucuronide methyl ester - Allows for the detection of E. coli in healthcare and food safety settings.

Masked antibiotics - Fosfomycin-beta-D-galactopyranoside sodium (MG179673) is a masked antibiotic which is specifically activated by the enzyme beta-galactosidase.

Our foundations of chemistry meets biology, enables Biosynth to continue to provide customers with unique and cutting edge technologies and products. As seen in our recent peer-reviewed publication, the Biosynth development team worked with veterinary researchers on a custom-made chromogenic medium using our innovative enzyme substrates. When selecting the most effective antibiotic protocols for dogs and cats with bacterial skin infections, the type of pathogen must first be identified. Our team demonstrated that our wide range of enzyme substrates could be tailored to the needs of clinicians and successfully detect different types of pathogens in a veterinary setting. The chromogenic substrates used in this study were: Aldol® 467 beta-D-galactopyranoside (A-4676_P00), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt trihydrate (B-7400), 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt trihydrate (B-7453), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside (B-7250), 1-O-Methyl-beta-D-glucuronic acid, sodium salt (M-3600). Read the full paper by clicking the link. 

Whether your applications be in research, medicine, therapeutics, food or veterinary, here at Biosynth we have a range of enzyme substrates to meet your needs. Find our list of enzyme substrates readily available in our catalog.  

References

Pala, L., Sirec, T., Spitz, U. (2020). Modified Enzyme Substrates for the Detection of Bacteria: A Review. Molecules, 25(16), 3690.