Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy

Ventura, Bartolomeo Della; Banchelli, Martina; Funari, Riccardo; Illiano, Anna; Angelis, Marella de; Taroni, Paola; Amoresano, Angela; Matteini, Paolo; Velotta, R.

doi:10.1039/c9an00443b

Cited by 40 publications

(51 citation statements)

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“…The adopted functionalization procedure was the photochemical immobilization technique (PIT) [ 31 ], a powerful and quick methodology based on an appropriate UV-activation of Abs, whose effectiveness was already confirmed in several application for biosensing [ 6 , 8 , 10 , 32 , 33 , 34 ]. It was demonstrated that this method leads at the same time to a strong (covalent) binding of Abs onto gold surfaces while orienting Abs with one fragment antigen-binding site (Fab) exposed to the solution.…”

Section: Methodsmentioning

confidence: 99%

“…Regarding the functionalization procedure, the photochemical immobilization technique (PIT) recently introduced by Della Ventura et al [ 31 ] not only was used for the first time to functionalize a ME material (coated with gold), but also the gold nanoflowers grown on the magnetic nanoparticles. This technique, based on a controlled UV-activation of Abs, has proven to be an effective [ 6 , 8 , 10 , 32 , 33 , 34 ] and competitive methodology since it is rapid and user-friendly and leads to strong (covalent) and conveniently oriented bonds of Abs on the sensor surfaces, without affecting the intrinsic selectivity of the antibodies.…”

Section: Introductionmentioning

confidence: 99%

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Core-Shell Magnetic Nanoparticles for Highly Sensitive Magnetoelastic Immunosensor

et al. 2020

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A magnetoelastic (ME) biosensor for wireless detection of analytes in liquid is described. The ME biosensor was tested against human IgG in the range 0–20 μg∙mL−1. The sensing elements, anti-human IgG produced in goat, were immobilized on the surface of the sensor by using a recently introduced photochemical immobilization technique (PIT), whereas a new amplification protocol exploiting gold coated magnetic nanoparticles (core-shell nanoparticles) is demonstrated to significantly enhance the sensitivity. The gold nanoflowers grown on the magnetic core allowed us to tether anti-human IgG to the nanoparticles to exploit the sandwich detection scheme. The experimental results show that the 6 mm × 1 mm × 30 μm ME biosensor with an amplification protocol that uses magnetic nanoparticles has a limit of detection (LOD) lower than 1 nM, works well in water, and has a rapid response time of few minutes. Therefore, the ME biosensor is very promising for real-time wireless detection of pathogens in liquids and for real life diagnostic purpose.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Core-Shell Magnetic Nanoparticles for Highly Sensitive Magnetoelastic Immunosensor

et al. 2020

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“…Gold NPs on the surface were functionalized with pan malaria antibodies (anti-PLDH) using PIT 39,49,50 . Such a technique consists of UV irradiation of Abs (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, due to their moderate long-time stability and the need to immobilize them both with the right orientation and high surface density, the accomplishment of a robust and effective Ab surface functionalization is still an open issue 38 . In this respect, the well-established photochemical immobilization technique (PIT) is a simple, fast and effective strategy to tether Abs onto gold surfaces with one fragment antigen-binding (Fab) exposed to the surrounding environment 39 . Besides antibodies, aptamers are nucleic acids which have been evolved to bind to targets through molecular evolution approaches as alternative class of bioreceptors.…”

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Ultrasensitive antibody-aptamer plasmonic biosensor for malaria biomarker detection in whole blood

et al. 2020

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Development of plasmonic biosensors combining reliability and ease of use is still a challenge. Gold nanoparticle arrays made by block copolymer micelle nanolithography (BCMN) stand out for their scalability, cost-effectiveness and tunable plasmonic properties, making them ideal substrates for fluorescence enhancement. Here, we describe a plasmon-enhanced fluorescence immunosensor for the specific and ultrasensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH)—a malaria marker—in whole blood. Analyte recognition is realized by oriented antibodies immobilized in a close-packed configuration via the photochemical immobilization technique (PIT), with a top bioreceptor of nucleic acid aptamers recognizing a different surface of PfLDH in a sandwich conformation. The combination of BCMN and PIT enabled maximum control over the nanoparticle size and lattice constant as well as the distance of the fluorophore from the sensing surface. The device achieved a limit of detection smaller than 1 pg/mL (<30 fM) with very high specificity without any sample pretreatment. This limit of detection is several orders of magnitude lower than that found in malaria rapid diagnostic tests or even commercial ELISA kits. Thanks to its overall dimensions, ease of use and high-throughput analysis, the device can be used as a substrate in automated multi-well plate readers and improve the efficiency of conventional fluorescence immunoassays.

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“…20 The Photochemical Immobilization Technique (PIT) is a surface functionalization procedure that only requires UV activation of the Abs and leads to a high density functionalized surface within minutes. 21,22 PIT has proven itself to be effective in tethering Abs upright not only on flat surfaces, [23][24][25][26] but also on AuNPs, which were used either to ballast small antigen 27 or to realize a colorimetric biosensor for detecting IgGs 28 and estradiol. 29 In the latter cases, the presence of the antigen was detected as a change in the absorbance that can be easily measured by a spectrophotometer or even by naked eye.…”

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Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs

Ventura¹,

Cennamo

Minopoli³

et al. 2020

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Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast and cheap tools to detect viral particles in biological material so to identify the people capable to spread the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles - AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope and membrane) - is redshifted in few minutes when mixed to a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a Real Time-PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of RT-PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open new perspective not only in the context of the current and possible future pandemics, but also in microbiology as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.

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Biosensor surface functionalization by a simple photochemical immobilization of antibodies: experimental characterization by mass spectrometry and surface enhanced Raman spectroscopy

Abstract: Thirty seconds of appropriate UV irradiation brings about specific thiol formation in IgGs that allows them to bind “side on” on a thiol reactive surface, thereby making such a photochemical approach a valuable tool for surface functionalization.

Cited by 40 publications

References 62 publications

Core-Shell Magnetic Nanoparticles for Highly Sensitive Magnetoelastic Immunosensor

Core-Shell Magnetic Nanoparticles for Highly Sensitive Magnetoelastic Immunosensor

Ultrasensitive antibody-aptamer plasmonic biosensor for malaria biomarker detection in whole blood

Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs

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