FK506 (tacrolimus) binding protein 12 (FKBP12) is involved in fundamental cellular processes as well as immunosuppression, neurodegenerative diseases and cancer. Hence, the ability to quickly and easily determine its levels is desirable. CLI caught up with Dr Pitruzzella, Dr Caminati and Prof. Cennamo to find out more about the point-of-care test that they have developed to assay FKBP12.

Can you tell us a bit about FKBP12, please?

FK506 binding protein (FKBP)-12 (encoded by the FKBP1A gene) is a 12-kDa cytosolic globular protein and the archetype of the immunophilin family. Immunophilins are highly conserved eukaryotic proteins characterized by the peptidyl-prolyl cis–trans isomerase (PPI) domain. FKBP12 possesses a single PPI domain, which acts as a chaperone in the folding and isomerization of proline-containing proteins. In our regular biology, it is involved in numerous metabolic and physiological processes, including the folding of developing polypeptide chains, protein transport, viral replication, calcium homeostasis. Additionally, it is involved in cell cycle regulation as it inhibits TGF-β receptor signalling. The absence of FKBP12 results in arrest of the cell cycle at the G1 phase.

Analysis of FKBP12 blood concentrations can plan effective and safe dosing levels for immunosuppressant drugs to prevent rejection of transplanted organs (Adobe Stock)

Why is FKBP12 an important biomarker?

The selective and rapid detection of FKBP12 is a key aspect in medicine, given its involvement in several diseases, making it a significant diagnostic and prognostic biomarker. FKBP12 was initially studied for its role in immunosuppression as it is a key protein that binds to immunosuppressive drugs such as tacrolimus (originally termed FK506) and rapamycin. Tacrolimus is used to treat patients

following organ transplant and also patients with autoimmune-related (T-cell-mediated) disorders, such as eczema and psoriasis. Tacrolimus binds to FKPB12 to create an FKBP12–tacrolimus complex, which inhibits calcineurin activation in T cells, so inhibiting T-cell signal transduction and IL-2 cytokine transcription. Hence, FKBP12 serves as a critical indicator for monitoring the risk of acute rejection, as its plasma concentrations are closely linked to acute rejection, and monitoring FKBP12 levels in the blood helps in the safe and effective dosing of these drugs to ensure sufficient pharmacological activity while minimizing toxic side effects. The importance of FKBP12 also extends to oncological diseases, where it is involved in tumorigenesis and in response to chemotherapy and radiotherapy, thereby increasing tumour cell sensitivity to chemotherapy-induced apoptosis. For example, low FKBP12 expression in breast cancer is associated with poor prognosis and resistance to certain treatments, making its detection crucial for guiding therapeutic strategies. FKBP12 has also been identified as a possible biomarker in neurodegenerative diseases, such as Parkinson’s disease, as its dysregulation and/or altered levels are strongly linked to the abnormal aggregation of proteins such as tau and alpha-synuclein, which are key features of the disease. Additionally, FKBP12 is associated with anti-self antibodies in autoimmune diseases. The involvement of FKBP12 in these processes and that it can be quickly, easily and reliably detected in biological fluids allows it to be a useful biomarker for monitoring disease progression, therapeutic efficacy and patient prognosis.

How is FKBP12 usually measured and why is there a need for a new test?

Current methods to detect low levels of FKBP12 employ general protein-detection techniques, including Western blotting, 2D gel electrophoresis, liquid chromatography-mass spectrometry, in situ hybridization, and ELISA (enzyme-linked immunosorbent assay). Despite its clinical relevance, there is currently a lack of rapid, low-cost methods for detecting traces of this biomarker. Conventional techniques, such as mass spectrometry-based methods, are often too complex and expensive, with detection limits in the nanomolar (nM) range. Although ELISA is reliable and sensitive (typically in the picomolar range), the process is generally lengthy and laborious, and it involves several complex procedures, such as biotin and streptavidin labelling, making it expensive and unsuitable for diagnostic applications. These limitations underscore the need for a low-cost, easy-to-use point-of-care test (POCT) that offers speed, small size, ultra-high sensitivity and selectivity to detect low levels of FKBP12 on-site without sample pretreatment.

Tacrolimus (Adobe Stock) FKBP12–tacrolimus complexes cause immunosuppression by inhibiting calcineurin activation in T cells.

How does your test work and what are its advantages?

The developed POCT is based on plasmonic phenomena implemented via a simple, low-cost and portable configuration that monitors disposable sensor chips (1 cm x 1 cm x 1 mm). The detection principle uses pollenbased nanostructures coated with gold nanofilms on a silica chip substrate. These gold nanostructures exhibit highly sensitive hybrid plasmonic phenomena, useful for monitoring analyte–receptor interactions. Therefore, the nanoplasmonic chip is functionalized with the synthetic receptor GPS-SH1, which selectively binds FKBP12. This receptor is combined with a spacer (C12-SH) to make an optimized self-assembled monolayer (SAM) on the nanoplasmonic surface. FKBP12 detection is performed by monitoring the shift in the plasmonic resonance wavelength, which occurs when receptor–analyte (FKBP12) binding changes the refractive index of the receptor layer in contact with the nanoplasmonic surface. This approach achieves ultra-high performance with a limit of detection (LOD) of 170 zM [zeptomolar (10-21) to attomolar (10-18) range], enabling single-molecule detection.

Key advantages include speed – requiring only 10 minutes of incubation – and low cost, thanks to the use of cheaper disposable chips produced with simpler and less expensive techniques. Moreover, the set-up (reader) is compact and requires low-cost equipment. Therefore, the proposed sensing approach offers significant advantages for the large-scale production of POCTs.

What are the potential future developments/applications for this technology?

This work highlights the capability of pollen-based plasmonic nanostructures combined with GPS-SH1 receptors to detect FKBP12 at the single-molecule level via POCTs. Considering the characteristics required of a POCT – ultra-low limit of detection, speed, low cost and portability – the main future prospects for this sensor system include advancing diagnostics, particularly by developing more easily accessible and highly sensitive diagnostic tools for FKBP12-associated diseases, such as cancer, neurodegenerative diseases, and autoimmune diseases. The test can also be used for therapeutic monitoring, allowing medical doctors to quickly and reliably assess the effectiveness of treatments and the patient’s prognosis. Ultimately, its simplicity and low-cost fabrication process make it highly suitable for widespread implementation in portable POC testing devices with disposable chips, thereby enabling more immediate and widespread clinical monitoring and diagnostics.

Bibliography
1. Tong M, Jiang Y. FK506-Binding Proteins and Their Diverse Functions. Curr Mol Pharmacol 2015;9(1):48–65 (https://doi.org/10.2174/1874467208666150519113541).
2. Caminati G, Procacci P, Menichetti S et al. A compound for the determination of the protein FKBP12 and a sensor unit comprising it. WO Patent 2021124269 A1. 2021, 24 June.
3. Cennamo N, Pasquardini L, Arcadio F, Zeni L. Pollen-based natural nanostructures to realize nanoplasmonic biochips for single molecule detection. Sens Actuators B Chem 2025;422:136404 (https://doi.org/10.1016/j.snb.2024.136404).
4. Marzano C, Pitruzzella R, Bartolini C et al. Point-of-care tests via pollen-based nanoplasmonic chips combined with a synthetic receptor for FKBP12 biomarker detection at a single-molecule level. ACS Appl Nano Mater 2025;8(40):19154–19166 (https://doi.org/10.1021/acsanm.5c02895).

The interviewees

Dr Rosalba Pitruzzella MD
PhD student

Department of Engineering, University of Campania Luigi Vanvitelli, Aversa 81031, Italy

Email address: rosalba.pitruzzella@unicampania.it

Professor Gabriella Caminati PhD
Assistant Professor in Physical Chemistry of Organized Systems

Department of Chemistry, University of Florence, Sesto Fiorentino 50019, Italy

Email address: gabriella.caminati@unifi.it

Professor Nunzio Cennamo PhD
Full Professor in Electronics

Department of Engineering, University of Campania Luigi Vanvitelli, Aversa 81031, Italy

Email address: nunzio.cennamo@unicampania.it