When an antibody is irradiated by Trylight... ...it covalently binds any noble metal surface, it stands upright.
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Promete is a small company that focuses its business on R&D and on bringing to the market new ideas from research to approach new business opportunities in selected niche markets. The company considers as main growth drivers the investment in IP, in the expertise in frontier applications and in high value-added and innovative products/services jointly developed through partnerships and collaborative actions with public and private entities.

In recent years PROMETE enhanced its R&D effort on biosensing through a tight collaboration with professor Raffaele Velotta (Physics Department of the University of Naples Federico II) for the development of sensors devices aimed at detecting foodborne pathogens. In this context, the surface functionalization procedure set up for binding oriented antibodies onto gold surfaces led us to contact Procom – a small company with expertise in UV lamps – with whom we designed and patented Trylight, an easy-to-use tool for carrying out the Photochemical Immobilization Technique. PIT is proving itself to be an effective way to tether antibodies on any thiol-reactive surface (e.g. gold), thus, the majority of the immunosensing platforms can take advantage of it.

TECHNICAL SHEET

Lamp type: PR7GL-5 253.7 .nm
Technical data
LAMP BODY: pure quartz of European origin
PIPE DIAMETER: 10 mm
FORM: U
LENGTH: total pin body excluding 45 mm
WIDTH: 40 mm
SUPPLY VOLTAGE: 12 Vdc
LAMP CURRENT: 170 mA
POWER OF SINGLE LAMP: 6 W
WORKING FREQUENCY: 35 KHz
UV EMISSION WAVELENGTH TYPE: PR7GL-5 254 nm
TOTAL UV INTENSITY ON THE CUVETTE: 0.3 W / cm2
OPERATING TEMPERATURE: -10 to 70 °C
HUMIDITY: max 95%
CONNECTION FEET: tungsten diam. 0.5 m

HOW IT WORKS

The Photochemical Immobilization Technique (PIT) is a functionalization method able to tether antibodies (Abs) on metal surfaces (such as gold or other bio-compatible metals) in a proper orientation that is with their binding sites well exposed to the environment.

This strategy is based on the selective photochemical reduction of disulfide bridges in immunoglobulins (Ig) produced by UV activation of near aromatic amino acid. Briefly, PIT is based on the selective photoreduction of disulphide bridges produced by the UV activation of the trp/cys-cys by means of a Trylight lamp.

The UV photon energy is adsorbed by tryptophan and transferred to near electrophilic species like the close cys-cys resulting in the cleavage of the disulphide bridge and in the formation of new reduced thiol (SH) groups able to bind. Considering that the triad of residues trp/cys-cys can be found in every Ig, the PIT is applicable in a wide range of fields. 

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FAQ
How long does it take Trylight to activate the antibodies (Ig)?
Only 30 s irradiation are enough to activate an antibody solution
What is the optimal concentration of antibodies to be used for irradiation?
Although 25 microg/mL is suggested in many applications, any concentration not exceeding 50 microg/mL can be used.
What kind of Ig can be immobilized by Trylight?
Many immunoglobulin G (IgG) have been successfully tethered to gold and silver surfaces by the photochemical immobilization technique (PIT), for which Trylight lends itself as an effective tool. Thus, while Trylight is expected to work for all IgGs, no evidence is reported so far about its application to other Igs.
How long IgGs holds activated once irradiated? How fast does one need to be in functionalizing the surface with the activated IgGs?
It has been demonstrated that after 5 minutes the irradiation is stopped, more than 70% of IgGs are still activated. Since the binding of activated IgGs with the surface is very effective, few minutes are typically enough to functionalize a surface of 1-100 mm2 by a simple fluidic circuit or even by drop casting.
What happens after 5 minutes of Trylight irradiation?
The specific thiols produced by Trylight begin to oxidate again and the disulfide bridges previously opened tend to close.
Is it possible to irradiate IgGs more than once?
Yes, but the thiol yield reduces its effectiveness each time by approximately 20%.
All the metal surfaces can be functionalized with IgGs?
While gold and silver have already been successfully tested, since the fundamental molecular mechanism of PIT relies on the selective thiol production in IgG by UV irradiation, any thiol reactive surface can be effectively passivated by using Trylight.
What kind of biosensor can be functionalized by PIT so that Trylight can be used?
Since the fundamental molecular mechanism of PIT relies on the selective thiol production in IgG by UV irradiation, any thiol reactive surface can be effectively passivated by using Trylight. Although biofunctionalization of metals like gold and silver have already has been successfully tested, it is expected that PIT and Trylight are of wider use.
Are commercial electrodes (e.g. screen-printed electrodes for electrochemistry) and chips (e.g. chips for Surface Plasmon Resonance) functionalizable “as are” or do they need a special treatment?
No special treatment of the surface is required other than usual washing when appropriate.
How long does it take Trylight to activate the antibodies (Ig)s?
Only 30 s irradiation are enough to activate an antibody solution
What is the optimal concentration of antibodies to be used for irradiation?
Although 25 microg/mL is suggested in many applications, any concentration not exceeding 50 microg/mL can be used.
What kind of Ig can be immobilized by Trylight?
Many immunoglobulin G (IgG) have been successfully tethered to gold and silver surfaces by the photochemical immobilization technique (PIT), for which Trylight lends itself as an effective tool. Thus, while Trylight is expected to work for all IgGs, no evidence is reported so far about its application to other Igs.
How long IgGs holds activated once irradiated? How fast does one need to be in functionalizing the surface with the activated IgGs?
It has been demonstrated that after 5 minutes the irradiation is stopped, more than 70% of IgGs are still activated. Since the binding of activated IgGs with the surface is very effective, few minutes are typically enough to functionalize a surface of 1-100 mm2 by a simple fluidic circuit or even by drop casting.
What happens after 5 minutes of Trylight irradiation?
The specific thiols produced by Trylight begin to oxidate again and the disulfide bridges previously opened tend to close.
Is it possible to irradiate IgGs more than once?
Yes, but the thiol yield reduces its effectiveness each time by approximately 20%.
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This web site has been realized under the project NEST that has received funding from the Regione Campania POR CAMPANIA FESR 2014-2020 ASSE III, OBIETTIVO SPECIFICO 3.4. AZIONE 3.4.2. Avviso pubblico per la concessione di contributi finalizzati al finanziamento di programmi di internazionalizzazione delle Micro e PMI campane. This web site contains material which is the copyright of PROMETE, and may not be reproduced or copied without permission, except as mandated by the Regione Campania for dissemination purposes. The content of the web site is the sole responsibility of the authors. The Regione Campania or its services cannot be held responsible for any use that may be made of the information it contains. However, the author(s) of this web site have taken any available measure to ensure that the information contained is accurate, consistent and lawful.