Search Results (39)

OoC systems employing human cells mirror the functionality of human organs and faithfully simulate their physiological microfluidic environment. Despite the potential of OoC technology in emulating tissue complexity, a significant gap persists in the continuous real-time monitoring of cellular behaviors and their responses to external stimuli, arising from the lack of biosensors integrated onto OoC microfluidic platforms. Addressing this limitation constitutes the primary objective of this study. By developing and incorporating biosensors onto a modular integrated OoC platform, we aim to enable the monitoring of changes taking place in the cellular environment under various stimuli in real time. An in-series modular integration of a biosensor array into an OoC platform is demonstrated herein, along with its potential to sustain human cell proliferation and accommodate the detection of IL-6, as an example of a mediator protein secreted as part of the immune response to inflammation. The implementation of commercially fabricated PCB components also addresses the issue of cost efficiency and manufacturing scaling-up of sensor-integrated OoCs. This advancement will not only enhance the accuracy and reliability of preclinical studies, but also pave the way for improved drug development and disease treatment. Full article

Aflatoxin M1 (AFM1) appears in the milk of animals that have consumed feed contaminated with aflatoxin B1. AFM1 presence in milk is regulated by the European Commission, which has set the maximum allowable limits for adult and infant consumption to 50 and 25 pg/mL, respectively. Here, a rapid and sensitive method for detecting AFM1 in milk based on an immersible silicon photonic chip is presented. The chip features two U-shaped silicon nitride waveguides formed as Mach–Zehnder interferometers. One interferometer is functionalized with AFM1–bovine serum albumin conjugate and the other with BSA to serve as a blank. The chip is connected to a broad-band white LED and a spectrophotometer by a bifurcated optical fiber and an assay is performed by immersing the chip in a mixture of milk with the anti-AFM1 antibody. Then, the chip is sequentially immersed in biotinylated anti-rabbit IgG antibody and streptavidin solutions for signal enhancement. The assay is completed in 20 min and the detection limit for AFM1 in undiluted milk is 20 pg/mL. Given its analytical performance and the absence of pumps and fluidics that lead to a compact instrument design, the proposed immunosensor is ideal for the on-site detection of AFM1 in milk samples. Full article

Aflatoxin M1 (AFM1) exposure through dairy products is associated with adverse health effects, including hepatotoxicity and carcinogenicity. Therefore, the AFM1 presence in milk and dairy products is strictly regulated. In this context, the current work focuses on the investigation of different competitive enzyme immunoassay configurations for the determination of AFM1 in milk with high sensitivity and short assay duration. Amongst the configurations tested, the one based on incubation of the anti-AFM1 specific antibody along with the calibrators/samples and a biotinylated conjugate of AFM1 with bovine serum albumin (BSA) in microwells coated with a secondary antibody provided a six-fold lower detection limit than the configuration involving immobilized AFM1-BSA conjugate and liquid phase antibody. The detection limit achieved was 5.0 pg/mL, with a dynamic range of up to 2.0 ng/mL. The assay was repeatable with intra- and inter-assay coefficients of variations lower than 3.2% and 6.5%, respectively, and accurate with recovery values from 87.5 to 108%. Moreover, the assay was completed in 1.5 h. The excellent analytical characteristics and short analysis time make the proposed assay suitable for use by the food industry. Furthermore, the proposed configuration could be employed to enhance the detection sensitivity of competitive immunoassays for other low-molecular-weight analytes. Full article

The enhanced and direct immobilization of the enzyme horseradish peroxidase on poly(methyl methacrylate) (PMMA) microchannel surfaces to create a miniaturized enzymatic reactor for the biocatalytic oxidation of phenols is demonstrated. Enzyme immobilization occurs by physical adsorption after oxygen plasma treatment, which micro-nanotextures the PMMA surfaces. A five-fold enhancement in immobilized enzyme activity was observed, attributed to the increased surface area and, therefore, to a higher quantity of immobilized enzymes compared to an untreated PMMA surface. The enzymatic reaction yield reached 75% using a flow rate of 2.0 μL/min for the reaction mixture. Additionally, the developed microreactor was reused more than 16 times without affecting the enzymatic conversion yield. These results demonstrate the potential of microchannels with plasma micro/nanotextured surfaces for the rapid and facile fabrication of microfluidic enzymatic microreactors with enhanced catalytic activity and stability. Full article

Survivin belongs to a family of proteins that promote cellular proliferation and inhibit cellular apoptosis. Its overexpression in various cancer types has led to its recognition as an important marker for cancer diagnosis and treatment. In this work, we compare two approaches for the immunochemical detection of survivin through surface-enhanced fluorescence or Raman spectroscopy using surfaces with nanowires decorated with silver nanoparticles in the form of dendrites or aggregates as immunoassays substrates. In both substrates, a two-step non-competitive immunoassay was developed using a pair of specific monoclonal antibodies, one for detection and the other for capture. The detection antibody was biotinylated and combined with streptavidin labeled with rhodamine for the detection of surface-enhanced fluorescence, while, for the detection via Raman spectroscopy, streptavidin labeled with peroxidase was used and the signal was obtained after the application of 3,3′,5,5′-tetramethylbenzidine (TMB) precipitating substrate. It was found that the substrate with the silver dendrites provided higher fluorescence signal intensity compared to the substrate with the silver aggregates, while the opposite was observed for the Raman signal. Thus, the best substrate was used for each detection method. A detection limit of 12.5 pg/mL was achieved with both detection approaches along with a linear dynamic range up to 500 pg/mL, enabling survivin determination in human serum samples from both healthy and ovarian cancer patients for cancer diagnosis and monitoring purposes. Full article

The quality and authenticity of milk are of paramount importance. Cow milk is more allergenic and less nutritious than ewe, goat, or donkey milk, which are often adulterated with cow milk due to their seasonal availability and higher prices. In this work, a silicon photonic dipstick sensor accommodating two U-shaped Mach–Zehnder Interferometers (MZIs) was employed for the label-free detection of the adulteration of ewe, goat, and donkey milk with cow milk. One of the two MZIs of the chip was modified with bovine κ-casein, while the other was modified with bovine serum albumin to serve as a blank. All assay steps were performed by immersion of the chip side where the MZIs are positioned into the reagent solutions, leading to a photonic dipstick immunosensor. Thus, the chip was first immersed in a mixture of milk with anti-bovine κ-casein antibody and then in a secondary antibody solution for signal enhancement. A limit of detection of 0.05% v/v cow milk in ewe, goat, or donkey milk was achieved in 12 min using a 50-times diluted sample. This fast, sensitive, and simple assay, without the need for sample pre-processing, microfluidics, or pumps, makes the developed sensor ideal for the detection of milk adulteration at the point of need. Full article

Oxidative stress is defined by an imbalance between the generation of reactive oxygen species and the biological system’s ability to neutralize them. This condition is commonly linked to various pathological conditions [¹]. Superoxide dismutase (SOD) is a widely used enzyme to assess oxidative stress, and various techniques have been developed for its detection in biological samples such as blood, urine, and saliva [²]. Surface-enhanced photoluminescence (PL) is a particularly sensitive method, offering minimal interference from the sample matrix [³]. In this work, silver nanostructured surfaces were implemented as substrates for the immunochemical determination of SOD in synthetic saliva through PL. The substrates were prepared using a single-step metal-assisted chemical etching method (MACE), resulting in the formation of silicon nanowires decorated with silver dendrites of approximately 1.5 μm in height [⁴]. For SOD detection, a three-step competitive immunoassay configuration was followed. Briefly, SOD was immobilized onto the substrates and then the functionalized substrates were incubated with mixtures of SOD with anti-SOD primary antibody, prepared either in assay buffer or synthetic saliva. Then, a solution of biotinylated anti-species specific antibody was added, followed by a reaction with streptavidin labelled with the fluorescent dye Rhodamine Red-X, and the signal was determined through an in-house developed optical set-up. The developed method presents similar or slightly lower sensitivity (detection limit 0.05 μg/mL) compared to the literature; however, it does not require labor-intensive sample pretreatment steps [⁵,⁶]. The aforementioned findings demonstrate the capability of the developed method to detect superoxide dismutase in natural saliva, in order to evaluate the oxidative stress status of an organism. Full article

Aflatoxin M1 (AFM1) is the hydroxylated form of Aflatoxin B1 (AFB1) and is expelled in the milk of both humans and animals following the consumption of AFB1-contaminated food. AFM1 has been categorized as a Group 1 carcinogen by the International Agency for Research on Cancer. Consequently, the European Commission has established a maximum allowable concentration of 50 pg/mL for AFM1 in dairy products and milk. Here, a rapid and sensitive approach for detecting AFM1 in bovine milk is presented. The analytical setup comprises a broad-band white LED, a spectrophotometer, and a silicon photonic probe, all interconnected by a bifurcated optical fiber [¹]. Additionally, a laptop powers the system and facilitates signal monitoring through specialized software. The silicon photonic probe is equipped with two Mach–Zehnder interferometers: one functionalized with AFM1-bovine serum albumin conjugate, and the other with bovine serum albumin to serve as a blank. The analysis involves immersing the probe directly into a mixture of anti-AFM1 antibodies and the sample, followed by sequential immersion into biotinylated anti-rabbit IgG antibody and streptavidin solutions. The entire assay process takes 12 min, and the limit of detection in undiluted milk is 20 pg/mL, below the EU maximum allowable limit of 50 pg/mL. The assay demonstrates accuracy, with %recovery values ranging from 87.5 to 112%, and repeatability, with intra/inter-assay coefficients of variation below 7.6%. Given its analytical performance and compact instrumentation, the proposed immunosensor proves to be an ideal solution for precise on-site determination of AFM1 in milk samples. Full article

Oxidative stress refers to the overproduction of reactive oxygen species and is often associated with numerous pathological conditions. Superoxide dismutase (SOD) is a widely used enzyme for evaluating oxidative stress, with numerous methods being developed for its detection in biological specimens like blood, urine, and saliva. In this study, a simple metal-assisted chemical etching method was employed for the fabrication of nanostructured silicon surfaces decorated with either silver dendrites or silver aggregates. Those surfaces were used as substrates for the immunochemical determination of SOD in synthetic saliva through surface-enhanced Raman spectroscopy (SERS) and surface-enhanced fluorescence (SEF). The immunoassay was based on a 3-step competitive assay format, which included, after the immunoreaction with the specific anti-SOD antibody, a reaction with a biotinylated secondary antibody and streptavidin. Streptavidin labeled with peroxidase was used in combination with a precipitating tetramethylbenzidine substrate for detection through SERS, whereas for SEF measurements, streptavidin labeled with the fluorescent dye Rhodamine Red-X was utilized. Both immunoassays were sensitive, with a detection limit of 0.01 μg/mL and a linear dynamic range from 0.03 to 3.3 μg/mL, enabling the evaluation of the oxidative stress status of an organism. Full article

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation. Full article

In this work, silicon nanowires were constructed by metal-assisted chemical etching and decorated with silver nanoparticles and used as substrates for the SERS determination of oxidative stress markers, namely glutathione, malondialdehyde and catalase. The assays were sensitive, with detection limits of 50 and 3.2 nM for glutathione and malondialdehyde, respectively, and 0.5 μg/mL for catalase, indicating the capability of the proposed substrates to be implemented for the determination of various oxidative stress markers. Full article

Nanostructured noble metal surfaces enhance the photoluminescence emitted by fluorescent molecules, permitting the development of highly sensitive fluorescence immunoassays. To this end, surfaces with silicon nanowires decorated with silver nanoparticles in the form of dendrites or aggregates were evaluated as substrates for the immunochemical detection of two ovarian cancer indicators, carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4). The substrates were prepared by metal-enhanced chemical etching of silicon wafers to create, in one step, silicon nanowires and silver nanoparticles on top of them. For both analytes, non-competitive immunoassays were developed using pairs of highly specific monoclonal antibodies, one for analyte capture on the substrate and the other for detection. In order to facilitate the identification of the immunocomplexes through a reaction with streptavidin labeled with Rhodamine Red-X, the detection antibodies were biotinylated. An in-house-developed optical set-up was used for photoluminescence signal measurements after assay completion. The detection limits achieved were 2.5 U/mL and 3.12 pM for CA125 and HE4, respectively, with linear dynamic ranges extending up to 500 U/mL for CA125 and up to 500 pM for HE4, covering the concentration ranges of both healthy and ovarian cancer patients. Thus, the proposed method could be implemented for the early diagnosis and/or prognosis and monitoring of ovarian cancer. Full article

Gonadotropin-releasing hormone (GnRH) is pivotal in regulating human reproduction and fertility through its specific receptors. Among these, gonadotropin-releasing hormone receptor type I (GnRHR I), which is a member of the G-protein-coupled receptor family, is expressed on the surface of both healthy and malignant cells. Its presence in cancer cells has positioned this receptor as a primary target for the development of novel anti-cancer agents. Moreover, the extensive regulatory functions of GnRH have underscored decapeptide as a prominent vehicle for targeted drug delivery, which is accomplished through the design of appropriate conjugates. On this basis, a rationally designed series of anthraquinone/mitoxantrone–GnRH conjugates (con1–con8) has been synthesized herein. Their in vitro binding affinities range from 0.06 to 3.42 nM, with six of them (con2–con7) demonstrating higher affinities for GnRH than the established drug leuprolide (0.64 nM). Among the mitoxantrone based GnRH conjugates, con3 and con7 show the highest affinities at 0.07 and 0.06 nM, respectively, while the disulfide bond present in the conjugates is found to be readily reduced by the thioredoxin (Trx) system. These findings are promising for further pharmacological evaluation of the synthesized conjugates with the prospect of performing future clinical studies. Full article

The COVID-19 pandemic has emphasized the urgent need for point-of-care methods suitable for the rapid and reliable diagnosis of viral infections. To address this demand, we report the rapid, label-free simultaneous determination of two SARS-CoV-2 proteins, namely, the nucleoprotein and the receptor binding domain peptide of S1 protein, by implementing a bioanalytical device based on Multi Area Reflectance Spectroscopy. Simultaneous detection of these two proteins is achieved by using silicon chips with adjacent areas of different silicon dioxide thickness on top, each of which is modified with an antibody specific to either the nucleoprotein or the receptor binding domain of SARS-CoV-2. Both areas were illuminated by a single probe that also collected the reflected light, directing it to a spectrometer. The online conversion of the combined reflection spectra from the two silicon dioxide areas into the respective adlayer thickness enabled real-time monitoring of immunoreactions taking place on the two areas. Several antibodies have been tested to define the pair, providing the higher specific signal following a non-competitive immunoassay format. Biotinylated secondary antibodies and streptavidin were used to enhance the specific signal. Both proteins were detected in less than 12 min, with detection limits of 1.0 ng/mL. The assays demonstrated high repeatability with intra- and inter-assay coefficients of variation lower than 10%. Moreover, the recovery of both proteins from spiked samples prepared in extraction buffer from a commercial self-test kit for SARS-CoV-2 collection from nasopharyngeal swabs ranged from 90.0 to 110%. The short assay duration in combination with the excellent analytical performance and the compact instrument size render the proposed device and assay suitable for point-of-care applications. Full article

Salmonella is one of the leading causes of foodborne illnesses worldwide, with poultry products being a major source of contamination. Thus, the detection of salmonella in commercial poultry products is crucial to minimize the effects on public health. Electrochemical sensors are promising tools for bacteria detection due to their sensitivity, simplicity, and potential for on-site analysis. In this work, a three-dimensional (3D) printed electrochemical immunosensor for the determination of Salmonella typhimurium in fresh chicken through a sandwich immunoassay employing biotinylated anti-S. typhimurium antibody followed by streptavidin labeled with Cd/Se ZnS quantum dots (QDs) is presented. The device features three carbon-black polylactic acid electrodes and a holder, and the quantification of S. typhimurium is performed by anodic stripping voltametric (ASV) determination of the Cd(II) released after acidic dissolution of the QDs. To enhance sensitivity, an electroplated bismuth film was deposited on the working electrode, achieving a detection limit of 5 cfu/mL in a total assay time of 25 min, whereas 5 h of sample pre-enrichment was required for the detection of 1 cfu/25 mL of chicken rinse and chicken broth. The method is accurate, with %recovery values ranging from 93.3 to 113% in fresh chicken samples, and repeatable with intra- and inter- assay coefficient of variations <2 and 5%, respectively, indicating the suitability of the proposed immunosensor for the detection of S. typhimurium at the point-of-need. Full article