- Autori:
-
Cenci, Lucia; Piotto, Chiara; Bettotti, Paolo; Maria Bossi, Alessandra
- Titolo:
-
Study on molecularly imprinted nanoparticle modified microplates for pseudo-ELISA assays
- Anno:
-
2018
- Tipologia prodotto:
-
Articolo in Rivista
- Tipologia ANVUR:
- Articolo su rivista
- Lingua:
-
Inglese
- Formato:
-
A Stampa
- Referee:
-
Sì
- Nome rivista:
- TALANTA
- ISSN Rivista:
- 0039-9140
- N° Volume:
-
178
- Intervallo pagine:
-
772-779
- Parole chiave:
-
Molecularly imprinted polymers; Nanoparticles; Hepcidin; Immunoassay; Molecularly imprinted sorbent assay; Pseudo-ELISA
- Breve descrizione dei contenuti:
- Nanosized Molecularly Imprinted Polymers (nanoMIPs) are designed artificial nanoreceptors with a predetermined selectivity and specificity for a given analyte, lately proposed as a replacement to antibodies in immunoassays. The nanoMIP-plate preparation based on nanoparticle adsorption was studied with the aim to rationally identify and discuss the critical points in the nanoMIP-assay development, in an example based on the iron homeostasis biomarker hepcidin and hepcidin-specific nanoMIPs (K-d = 9 nM). Plates were prepared by deposition and drying of nanoMIP (0.5-4 mu g/well), or by nanoMIPs co-depositions (proteins, PVA). Rehydration ( > 1 h) of dry nanoMIP-plates showed the reconstitution of the imprinted binding sites. NanoMIP-plate mechanical stresses (several washings; pipetting) caused nanoMIP desorption (similar to 90%). After 10 washes the quantity of nanoMIP was 0.2 mu g/well, the imprinted binding sites were similar to 270 fmol/well, their accessibility the 92%. Co-depositions resulted in higher amount of adsorbed nanomaterial (1.2 mu g/well), but low accessibility of the imprinted binding sites (2-47%). Tested in a competitive sequential assay, using as competitor horseradish peroxidase conjugate to hepcidin, the nanoMIP-plate permitted to determine hepcidin in serum samples, yet with a narrow dynamic range of response (0.9-10 nM). Critical points in the assay were: the instability of the nanoMIP adsorption, which lead to the progressive loss of binding sites/well, and the affinity of the nanoMIP for the analyte (K-d = 9 nM), which corresponds to kinetics dissociation constants on the time-scale of the washing lengths (minutes), thus compatible with the release of the bound hepcidin during the washings. The found limits set the conditions to develop a successful nanoMIP-assay: (i) stable microplate derivatization; (ii) maximized number of imprinted binding sites/well; (iii) nanoMIP/analyte equilibrium not perturbed on the time scale of the minutes (i.e. K-d similar to pM).
- Id prodotto:
-
99968
- Handle IRIS:
-
11562/972005
- ultima modifica:
-
2 dicembre 2022
- Citazione bibliografica:
-
Cenci, Lucia; Piotto, Chiara; Bettotti, Paolo; Maria Bossi, Alessandra,
Study on molecularly imprinted nanoparticle modified microplates for pseudo-ELISA assays
«TALANTA»
, vol.
178
,
2018
,
pp. 772-779
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