Sandro Spagnolo

Myelin basic protein (MBP) is an important biomarker of demyelination and is elevated in multiple sclerosis and other neuroinflammatory disorders. We developed an electrochemical and quartz crystal microbalance with dissipation monitoring (QCM-D) sensor for MBP detection combining a DNA aptamer with antifouling surface chemistry. Gold electrodes were modified with mixed HS-MEG-COOH/DTT self-assembled monolayers to minimize nonspecific adsorption. MBP-specific amino-modified DNA aptamers were covalently immobilized at the monolayer surface by carbodiimide chemistry. Differential pulse voltammetry (DPV) was used to monitor aptamer–MBP binding, enabling quantitative detection from 0.5 ng/mL to 400 ng/mL. The limit of detection (LOD) was similar in buffer (2.74 ng/mL (DPV) and 4.14 ng/mL (QCM-D)) and in artificial cerebrospinal fluid (aCSF) (6.36 ng/mL (DPV) and 6.87 ng/mL (QCM-D)).

Myelin basic protein (MBP) is released into cerebrospinal fluid during demyelination and is therefore a promising biomarker for the detection of neurodegenerative diseases such as multiple sclerosis. For the label-free detection of MBP, we developed an antifouling aptasensor based on thickness shear mode sensing. DNA aptamers were immobilized on gold-coated piezoelectric crystals functionalized with an engineered antifouling self-assembled monolayer based on 3-(2-mercaptoethoxy)propanoic acid (HS-MEG-COOH) combined with either 2-(2-mercaptoethoxy)ethanol (HS-MEG-OH) or dithiothreitol (DTT) as spacers. The binding of recombinant human MBP to the aptamer was confirmed with a corresponding decrease in resonance frequency in both phosphate-buffered saline (PBS) and artificial cerebrospinal fluid (aCSF). The aptasensor achieved a limit of detection of 11 ng mL⁻¹ in PBS and 2 ng mL⁻¹ in aCSF. PBS measurements were primarily applied to determine the aptasensor functionality, whereas experiments in aCSF evaluated MBP detection at concentrations relevant to reported levels in biological CSF. Selectivity was assessed with aptamer-free surfaces, cytochrome c, and human serum albumin (HSA) with much lower responses, while concentration-dependent MBP detection was maintained in aCSF despite the increased complexity of the matrix. The antifouling performance of the modified sensing surface was further demonstrated in aCSF by the significantly reduced frequency and dissipation responses compared to bare gold. With future validation in human cerebrospinal fluid (CSF) samples, the developed biosensor has the potential to enable sensitive, label-free detection of MBP in CSF for the identification and monitoring of neurodegenerative diseases.

Penicillin G (PEN) is a widely used antibiotic for treating microbial infections. However, its extensive use in veterinary medicine can lead to accumulation in animal-derived products, particularly milk and meat. This highlights the urgent need for rapid and sensitive antibiotic detection methods. In this study, we employed DNA aptamers for the detection of PEN and for the analysis of aptamer specificity using a combined approach based on quartz crystal microbalance with dissipation monitoring (QCM-D) and localized surface plasmon resonance (LSPR). QCM-D measures changes in resonant frequency, Δf, and dissipation, ΔD, while LSPR monitors wavelength shifts in the extinction spectra corresponding to changes at the surface of gold nanoparticles (AuNPs). Thiolated aptamers were chemisorbed onto the surface of AuNPs with a diameter of 80 nm. In the presence of PEN, a redshift in the extinction spectra and a decrease in resonant frequency were observed, accompanied by an increase in dissipation due to surface viscosity effects. Significant changes in both acoustic and LSPR signals were observed at PEN concentrations as low as 1 nM. The limits of detection (LOD) for PEN, determined by QCM-D (3.0 nM, or 1.05 ng/mL)) and LSPR (3.1 nM, or 1.09 ng/mL), were similar and both were lower than the maximum residue limit (MRL) for PEN established by the EU (4 ng/mL).

A biosensor was designed for detecting Pseudomonas aeruginosa (P. aeruginosa) bacteria in whole milk samples. The sensing layer involved the antifouling linking molecule 3-(2-mercaptoethanoxy)propanoic acid (HS-MEG-COOH), which was covalently linked to an aptamer for binding P. aeruginosa. The aptasensor uses the thickness shear mode (TSM) system for mass-sensitive acoustic sensing of the bacterium. High concentrations (10⁵ CFU mL⁻¹) of nonspecific bacteria, E. coli, S. aureus, and L. acidophilus, were tested with the aptasensor and caused negligible frequency shifts compared to P. aeruginosa. The aptasensor has high selectivity for P. aeruginosa, with an extrapolated limit of detection (LOD) of 86 CFU mL⁻¹ in phosphate-buffered saline (PBS) and 157 CFU mL⁻¹ in milk. To improve the sensitivity of the sensor, gold nanoparticles (AuNPs) were functionalized with the same aptamer for P. aeruginosa and flowed through the sensor following bacteria, reducing the extrapolated LOD to 68 CFU mL⁻¹ in PBS and 46 CFU mL⁻¹ in milk. The frequency variations in the aptasensor are proportional to various concentrations of P. aeruginosa (10²–10⁵ CFU mL⁻¹) with and without AuNPs, respectively. The low and rapid mass-sensitive detection demonstrates the ability of the aptasensor to quantitatively identify bacterial contamination in buffer and milk.

Ovarian cancer (OC) must be detected in its early stages when the mortality rate is the lowest to provide patients with the best chance of survival. Lysophosphatidic acid (LPA) is a critical OC biomarker since its levels are elevated across all stages and increase with disease progression. This paper presents an LPA assay based on a thickness shear mode acoustic sensor with dissipation monitoring that involves a new thiol molecule 3-(2-mercaptoethanoxy)propanoic acid (HS-MEG-COOH). HS-MEG-COOH is an antifouling linker that provides (a) antifouling properties for gold substrates and (b) linking ability via its terminal carboxylic acid functional group. The antifouling ability of HS-MEG-COOH was tested in whole human serum. The new molecule was applied to the LPA assay in conjunction with a spacer molecule, 2-(2-mercaptoethoxy)ethan-1-ol (HS-MEG-OH), in a 1:1 v/v ratio. HS-MEG-COOH was covalently linked to gelsolin–actin, a protein complex probe that dissociates due to LPA-binding. LPA was detected in phosphate-buffered saline and undiluted human serum and achieved a low limit of detection (1.0 and 0.7 μM, respectively) which was below the concentration of LPA in healthy individuals. The antifouling properties of HS-MEG-COOH and the detection of LPA demonstrate the ability of the sensor to successfully identify the early-stage OC biomarker in undiluted human serum.

Electrochemical biosensors are valued for their sensitivity and selectivity in detecting biological molecules. Having the advantage of generating signals that can be directly or indirectly proportional to the concentration of the target analyte, these biosensors can achieve specificity by utilizing a specific biorecognition surface designed to recognize the target molecule. Electrochemical biosensors have garnered substantial attention, as they can be used to fabricate compact, cost-effective devices, making them promising candidates for point-of-care testing (POCT) devices. This study introduces a label-free electrochemical biosensor employing a gold screen-printed electrode (SPE) to detect lysophosphatidic acid (LPA), a potential early ovarian cancer biomarker. We employed the gelsolin–actin system, previously introduced by our group, in combination with fluorescence spectrometry, as a biorecognition element to detect LPA. By immobilizing a gelsolin–actin complex on an SPE, we were able to quantify changes in current intensity using cyclic voltammetry and differential pulse voltammetry, which was directly proportional to the LPA concentration in the solution. Our results demonstrate the high sensitivity of the developed biosensor for detecting LPA in goat serum, with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.9 µM and 2.76 µM, respectively, highlighting its potential as a promising tool for early-stage diagnosis of ovarian cancer.

The overall 5-year survival rate of ovarian cancer (OC) is generally low as the disease is often diagnosed at an advanced stage of progression. To save lives, OC must be identified in its early stages when treatment is most effective. Early-stage OC causes the upregulation of lysophosphatidic acid (LPA), making the molecule a promising biomarker for early-stage detection. An LPA assay can additionally stage the disease since LPA levels increase with OC progression. This work presents two methods that demonstrate the prospective application for detecting LPA: the electromagnetic piezoelectric acoustic sensor (EMPAS) and a chemiluminescence-based iron oxide nanoparticle (IONP) approach. Both methods incorporate the protein complex gelsolin–actin, which enables testing for detection of the biomarker as the binding of LPA to the complex results in the separation of gelsolin from actin. The EMPAS was characterized with contact angle goniometry and atomic force microscopy, while gelsolin–actin-functionalized IONPs were characterized with transmission electron microscopy and Fourier transform infrared spectroscopy. In addition to characterization, LPA detection was demonstrated as a proof-of-concept in Milli-Q water, buffer, or human serum, highlighting various LPA assays that can be developed for the early-stage detection of OC.

Cytochrome c (cyt c) is an important indicator of cell apoptosis and can, therefore, be used for the diagnosis of cancer. We performed a comparative analysis of cyt c detection on the surface of lipid films or a monolayer of 11-mercaptoundecanoic acid (MUA) with immobilized specific or nonspecific DNA aptamers. A quartz crystal microbalance with dissipation monitoring (QCM-D) in a multiharmonic mode was used to study the interaction of cyt c with various surfaces. For this purpose, changes in the resonant frequency, Δf, and dissipation, ΔD, were determined. The strongest interaction of cyt c was observed with sensors based on specific DNA aptamers that were accompanied by a decrease in frequency and an increase in dissipation. The limit of detection (LOD) for this aptasensor was established as 2.89 ± 0.12 nM. The interaction of cyt c with supported lipid films also resulted in a decrease in resonant frequency, but significant changes occurred only in the µM concentration range of cyt c. Changes in dissipation were much lower in comparison with aptamer-based surfaces, which suggests a weaker contribution of cyt c adsorption to the viscosity.

Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination of food. In this work, an aptasensor based on a thickness shear mode acoustic method (TSM) with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow’s milk. The frequency variation and dissipation data demonstrated the correct immobilization of the components. The analysis of viscoelastic properties suggests that DNA aptamers bind to the surface in a non-dense manner, which favors the binding with bacteria. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the sensor’s antifouling properties, which is based on 3-dithiothreitol propanoic acid (DTT_COOH) antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) quartz crystals, the sensitivity of the sensor’s antifouling in milk improved by about 82–96%. The excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow’s milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.

We analyzed the possibility of the detection of cytochrome c (cyt c) being physically adsorbed on lipid films or covalently bounded to 11-mercapto-1-undecanoic acid (MUA) chemisorbed on the gold layer using quartz crystal microbalance with dissipation monitoring (QCM-D). The negatively charged lipid film composed of a mixture of zwitterionic DMPC and negatively charged DMPG phospholipids at a molar ratio of 1:1 allowed the formation of a stable cyt c layer. Addition of DNA aptamers specific to cyt c, however, resulted in removal of cyt c from the surface. The interaction of cyt c with the lipid film and its removal by DNA aptamers were accompanied by changes in viscoelastic properties evaluated using the Kelvin–Voigt model. Cyt c covalently bound to MUA also provided a stable protein layer already at its relatively low concentrations (0.5 μM). A decrease in the resonant frequency following the addition of gold nanowires (AuNWs) modified by DNA aptamers was observed. The interaction of aptamers with cyt c on the surface can be a combination of specific and non-specific interactions due to electrostatic forces between negatively charged DNA aptamers and positively charged cyt c.