The use of pesticides has been continuously increasing worldwide since their introduction in the global market, with the aim of fulfilling the needs of the growing population. While their monitoring in the environment has been thoroughly addressed, the bioaccumulation of pesticides in the human body is an ongoing challenge, due to the complexity of the biological matrices in which pesticides tend to accumulate (e.g., urine, blood, sweat, and more). Most of the developed strategies rely on time-consuming sample preparation or expensive and polluting reagents for the construction of the sensing platform. In the view of developing a green alternative device to handle pesticide analysis in biofluids, we designed an enzymatic paper-based electrochemical sensor for glyphosate (GLY) detection in human urine. The pesticide quantification is obtained by measuring the grade of enzyme inhibition by both GLY and uric acid (UA), the latter being one of the principal components of human urine. GLY detection is carried out by measuring the initial and residual enzymatic activity in chronoamperometric tests, taking into consideration the contribution of UA to the inhibition. In addition to the specific application here described, this work aims at providing an emblematic example of how to design green sensing solutions for addressing analytical challenges related to health control, delivering low-impact methods based on sustainable materials (e.g., paper), non-toxic reagents, and minimal waste production while ensuring competitive analytical performances.

Heavy metals ions (HMI) are micropollutants that represent a growing environmental problem, as they have influenced various components of the environment, such as terrestrial and aquatic biota. Among HMI, lead and cadmium are highly toxic, and even small doses can lead to harmful effects on human health. Thus, rapid methods to detect these metals in multi-matrices are urgently required. Here, an electrochemical sensor screen-printed onto a flexible substrate has been coupled with a paper-based platform for the determination of HMI in clinical, environmental and food matrices have been developed. The bismuth film-based flexible device has been optimized and it has been able to detect cadmium and lead, respectively, down to the detection limit of 1.3 and 2 ppb. The use of chromatographic paper has allowed to improve the sensitivity towards the detection of HMI, because of the porosity that allowed to pre-concentrate species. The combination of this platform with a paper-based one has allowed to enhance the sensitivity of the whole device, with a detection limit of 0.3 and 0.5 ppb, respectively, to cadmium and lead, and offers the possibility to tune the sensitivity according to needs, e.g., improving the number of pre-concentration steps. The electrochemical sensor was evaluated in drinking water, mussel and blood serum, demonstrating how these hybrid polyester-paper electrochemical strips can significantly lower the time and costs for on-site measurements, through analytical methods of simple use. The accuracy has been evaluated by comparison with ICP-MS measurements, giving satisfactory results.

DOI 10.1149/1945-7111/ac5c98

Three-dimensional (3D) printing has gained significant attention from industry and research laboratories, as it empowers the end user with the freedom to create in-house and on-demand specialized electrochemical systems adapted to immediate bioanalytical needs. Fused deposition modeling (FDM) is based on the CAD design of the sensor and its printing from thermoplastic filaments. FDM presents advantageous features such as low-cost portable printers, ease of operation, flexibility in the design, design transferability, and thus, it can complement and in some cases replace, existing fabrication technologies [1,2,3].

This presentation will discuss our recent developments of 3D-printed integrated chips and their applications to electrochemical biosensing [4,5,6]. The devices are printed from specific filaments (conductive and non-conductive) at different sizes and shapes, by a dual extruder 3D printer, in order to fit to the demands of various applications. More specifically, integrated all-3D-printed electrochemical microtitration wells (e-wells) for the in-situ and micro-volume quantum dot based bioassays will be described. Besides, a 3D printed 4-electrode biochip for the enzymatic simultaneous determination of two biomarkers and a 3D printed wearable glucose monitoring device in the form of an electrochemical ring (e-ring) will be presented.

References

1. A. Abdalla and B. A. Patel, Curr. Opin. Electrochem, 20 (2020) 78–81.
2. E. J. Carrasco-Correa, E. F. Simó-Alfonso, J. M. Herrero-Martínez, M. Miró, TrAC - Trends Anal. Chem., 136 (2021) 116177.
3. V. Katseli, A. Economou, C. Kokkinos, Electrochem. Commun. 103 (2019) 100–103.
4. V. Katseli, M. Angelopoulou, C. Kokkinos, Adv. Funct. Mater., (2021) 2102459.
5. V. Katseli, A. Economou, C. Kokkinos, Anal. Chem. 93 (2021) 3331−3336.
6. E. Koukouviti, C. Kokkinos, Anal. Chim. Acta 1186 (2021) 339114

Ochratoxin A (OTA) is a mycotoxin produced by several fungal species and various studies have shown that OTA can cause several adverse health effects to animals and humans through its consumption in contaminated plant foods such as coffee, beer, wine, corn, wheat, oats and vegetables [1]. OTA has been shown to be nephrotoxic, teratogenic, immunotoxic, and carcinogenic in human health. In particular, the International Agency for Research on Cancer has classified OTA as a group 2B carcinogen [2]. Due to the toxicity of OTA , the European Union has set maximum limits (MLs) for OTA in foods in the range of 0.5–10 μg kg⁻¹ [2]. Therefore, the development of a cheap, sensitive and rapid method for OTA detection in plant-based foods is essential.

The detection of OTA in food is mostly based on chromatographic techniques, which although powerful , require expensive equipment, trained personel and complex sample preparation [1,2]. Enzyme-linked immunosorbent assay s(ELISA) and immunochromatographic assays are portable, convenient and simple but require expensive and unstable antibodies and normally [3,4]. On the contrary aptamer-based biosensors employ relative inexpensive and stable single stranded oligonucleotides as biorecognition elements, which makes them ideal for rapid on-site detection of OTA [2,5], especially when combined with smartphone-based detection [6].

In this work, we describe a simple, portable and cost-efficient lateral flow biosensor for OTA. The biosensor utilizes an OTA-specific aptamer for biorecognition and is based on a lateral flow assay using a device consisting of a sample pad, a conjugate pad, a test and a control zone, as well as an absorbent pad. The conjugate PAD is loaded with OTA aptamer-AuNPs congugates while the test and control zones are loaded with a specific and a universal probe, respectively. The principle of the assay is that the OTA present in the sample combines with the OTA aptamer-AuNPs congugates and prevents the interaction between the specific probe in the test line and the OTA aptamer-AuNPs congugates; therefore, the intensity of the test line decreases as the concentration of OTA in the sample increases. Quantification of OTA is performed by reflectance calorimetry using a smartphone and image analysis. All the parameters of the assay were investigated in detail and the analytical features were established.

References

[1] Atumo S (2020) A Review of Ochratoxin A Occurrence, Condition for the Formation and Analytical Methods. Int J Agric Sc Food Technol 6(2): 180-185. DOI: 10.17352/2455-815X.000071

[2] Chen, X., Gao, D., Sun, F. et al. Nanomaterial-based aptamer biosensors for ochratoxin A detection: a review. Anal Bioanal Chem 414, 2953–2969 (2022). https://doi.org/10.1007/s00216-022-03960-5

[3] Meulenberg EP. Immunochemical methods for ochratoxin A detection: a review. Toxins (Basel). 2012 Apr;4(4):244-66. doi: 10.3390/toxins4040244. Epub 2012 Apr 13. PMID: 22606375; PMCID: PMC3347002

[4] Bazin I, Nabais E, Lopez-Ferber M. Rapid visual tests: fast and reliable detection of ochratoxin A. Toxins. 2010 Sep;2(9):2230-2241. DOI: 10.3390/toxins2092230

[5] Ha, T.H. Recent Advances for the Detection of Ochratoxin A. Toxins 2015, 7, 5276-5300. https://doi.org/10.3390/toxins7124882

[6] Madrid RE, Ashur Ramallo F, Barraza DE, Chaile RE. Smartphone-Based Biosensor Devices for Healthcare: Technologies, Trends, and Adoption by End-Users. Bioengineering (Basel). 2022 Mar 1;9(3):101. doi: 10.3390/bioengineering9030101

Biocidal disinfectants are used daily throughout the food chain to limit the development of undesirable microorganisms present in the environment or on surfaces in contact with foodstuffs or animal feed.

The presence of residues of these biocidal products is an issue for human health especially if they are not completely removed during rinsing operations.

Today, we aim to develop innovative electrochemical biosensors as powerful analytical methods to detect these residues of biocides.

Enzyme-based biosensors are known to be more sensitive and selective. So far, only one conductometric sensor is known at present for the detection of surfactants [1]. In this light, we suggested applying the acetylcholinesterase-based voltammetric biosensor for inhibitory analysis of quaternary ammonium compounds (QACs).

In this work, we elaborated an enzymatic sensor based on bionanomaterials film consisting of a fil of carboxylic acid functionalized multi-walled carbon nanotubes (c-MWCNT) modified with the acetylcholinesterase enzyme. In addition to their mechanical stability and good electric conductivity, the c-MWCNT in particular display a large number of binding sites available for enzyme immobilization. These cholinesterase-based sensors are very sensitive and allow for reaching low limits of detection.

Cyclic voltammetry was used to characterize the sensing film deposited onto the surface of electrodes such as glassy carbon and gold electrodes (Au250AT, Au250BT).

The acetylcholinesterase sensors that exhibited good repeatability and stability were applied to the detection of residues of QACs in milk samples.

[1] DOI 10.1088/0957-0233/23/6/065801

The aim of the present work is the preliminary investigation of a platform for the optimal implementation of chemiluminescence (CL) based biosensing. Among all the optical strategies for biosensing, CL offers many positive features, such as high sensitivity of detection even in low volumes, no need for any external light source and simple instrumentation required for its measurement, which make it particularly suited for the development of ultrasensitive assays in a portable format for point-of-care (POC) settings. Nevertheless, the analytical performance of most of these portable devices is mainly limited by an inefficient optical coupling between the biosensor compartment where photon emission occurs (e.g., microfluidic chip) and the photodetector. While the principally exploited strategy is to place the CL chamber as close as possible to the photon detector, this leads to an ideal maximum photon collection efficiency of 50%, which however is usually much lower, due to several factors, such as reflection phenomena at interfaces between different materials. Therefore, a large fraction of emitted photons is lost, leading to lower assay sensitivity. Very recently, there was a growing interest in optical fiber CL-based biosensors where the fiber plays a crucial role both in the transduction and transportation of the generated light. Despite the recent interesting results on CL optical fiber immunosensors, it must be emphasized that an accurate and deep analysis of the optical properties of the interaction between the CL signal and the dielectric guide has not been fully addressed so far. The aim of this work is the realization of an optimized optical fiber CL biosensing system in which the reaction occurs very close to the external lateral surface of an optical fiber core. Preliminary data were obtained by a modified multimode fiber inserted in a capillary used to monitor a CL signal generated after an ovalbumin immunoassay.

Acknowledgments. This work was supported by the Italian Ministry for University and Research in the framework of “Bando PRIN 2017”, project number 2017YER72K.

Lysozyme is a glycoside hydrolase enzyme commonly found in body fluids such as saliva, tears, and milk [1]. Lysozyme has an important role in food industry due to its antibacterial activity. It is used as a stabilizer, shelf-life enhancer, and preventer of butyric acid formation [2].

Lysozyme is a potentially allergenic substance [3]. Even trace amounts of Lysozyme in foods can trigger adverse reactions in the immune system in sensitive individuals. Additionally, it is considered as one of the five main allergenic proteins found in the egg of the domestic chicken (Gallus domesticus) [4]. Therefore, detecting Lysozyme in food, beverage, and alcohol samples is critical to avoiding its allergenic effect.

Aptamers can bind to their targets with high affinity due to their three-dimensional conformation, similar to antibodies [6]. Molecular Imprinted Polymers (MIP) have high affinity, and superior selectivity compared to their counterparts; they are resistant to harsh conditions such as temperature, pressure, and pH changes. An important advantage that a MIP has in analysis applications is its molecular design or imprinting specificity Integration of MIP with aptamer (aptamer-MIP) can provide a hybrid system with the excellent binding specification with analyte molecules, containing the advantages of both methods.

In this study, an aptamer-MIP-based biosensor was prepared for the Lysozyme determination. Screen-printed electrodes (SPEs) (DRP-110) were modified with gold nanoparticles (AuNPs) and graphene oxide (GO) to increase conductivity and surface area. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) were used in the optimization and surface characterization of the study. The developed Analytical applicability of the prepared aptamer-MIP biosensor was tested in different real samples.

Molecularly imprinted polymers (MIPs) are artificial biomimetic materials attracting increasing attention due to ease of synthesis combined with strength, robustness and molecular recognition capabilities on a pair with those of biological elements (e.g. antibodies and enzymes) [1]. As “antibody mimics”, MIPs are used in a multitude of fields, and the number of applications is constantly increasing due to the improvement and development of new synthetic approaches. In this context, photostructuring of MIPs is particularly attractive because of the possibility of tightly controlling their features in terms of size, morphology and thickness. Here, we propose to take advantage of photo-controlled radical polymerisation, for the deposition of MIPs on nanostructured porous silicon (pSi), with high aspect ratio (100) and columnar pores with size around 50 nm, used as interferometer. PSi has been increasingly exploited in bio/chemosensing due to its huge specific surface, straightforward fabrication and low cost, which allows mass production of cheap biosensors for point-of-care application to be envisioned. In the present work MIPs against propranolol as model target were synthesized within nanoporous silicon under visible light. A homogeneous thin layer deposition was achieved on pSi, as evidenced by UV-Vis reflectance spectroscopy. The resulting sensor was challenged toward propranolol detection and preliminary results indicated good linearity in the concentration range from 5 to 100 µM with a LOD of 3 µM. Propranolol detection tests performed in tap water confirm the ability of the sensor to detect the target in real matrices. Moreover, detection tests using metoprolol, atenolol and timolol (other b-blockers) as interfering molecules demonstrate a good selectivity of the developed sensor.

Acknowledgements

This work was partially funded by the European Union Horizon Europe programme under grant agreement No 101046946 (RESORB).

References

1. Mazzotta E, Di Giulio T, Malitesta C (2022) Electrochemical sensing of macromolecules based on molecularly imprinted polymers: challenges, successful strategies, and opportunities. Anal Bioanal Chem. https://doi.org/10.1007/S00216-022-03981-0

Identifying a suitable textile electrode that would be durable and assist in recording high bio-signal quality is crucial in the production of medical devices. Therefore, this study aimed to compare silver-plated-polyamide-embroidered cotton (SPEC), copper-nickel-plated polyester (CNP), and stainless-steel-fabric (SSF) dry textile electromyography (EMG) electrodes through principal component analysis (PCA). Each electrode was also compared against standard silver/silver chloride (Ag/AgCl) gel electrodes. The output was analyzed based on multi variables extracted from EMG features such as root mean square (RMS) voltage, average rectified value (ARV) voltage, kurtosis, and skewness using activation of the bicep, and tibialis anterior muscles group. The SSF electrode outperformed CNP and SPEC electrodes. Without applying any boundary conditions, each textile electrode has signal-to-noise ratio (SNR) values comparable to the standard electrode. The SNR values were 24.38 dB, 17.72 dB, 15.55 dB, and 13.30 dB, for Ag/AgCl, SSF, CNP and SPEC electrodes, respectively. The performance of all the conductive textile electrodes was comparable to that of Ag/AgCl. However, the gel electrode requires skin preparation and has short-term stability, whereas textile electrode materials last longer and can be used for biological signal monitoring at home without the assistance of medical professionals.

Lateral flow assay (LFA) is a powerful tool for rapid screening of target biomarker. With colorimetric analysis, LFA can be a quantitative tool as well as qualitative one, which allows LFA to be a good point of care testing device. However, the concentration of several biomarkers for disease diagnosis is far lower than the detection limit of colorimetric LFA. Although various labels, such as fluorescent beads, quantum dots, and upconverting nanoparticles, have been applied to LFA for better sensitivity, the detection of a skeletal muscle protein, called cardiac troponin-I (cTnI), needs picogram-level sensitivity and high selectivity.

To address this issue, surface enhanced Raman spectroscopy (SERS) was applied to LFA flatform to achieve ultrasensitive detection of cTnI. Au@Ag core-shell nanoparticle was introduced as a SERS tag to increase Raman intensity than bare gold nanoparticle. The LOD of colorimetric and SERS analysis of cTnI was 5 ng/ml and 50 pg/ml, which was about 100-fold increment by applying SERS and Au@Ag NPs. Also, selectivity to cTnI against other blood proteins such as human serum albumin and gamma globulin was verified. As a result, SERS-LFA is a powerful platform for ultrasensitive detection of picogram-level of biomarkers such as cTnI.