An ultrasensitive and disposable electrochemical aptasensor for prostate-specific antigen (PSA) detection in real serum samples.

In this study, we constructed a disposable indium tin oxide polyethylene terephthalate film (ITO-PET)-based electrochemical aptasensor for analyzing prostate-specific antigen (PSA), one of the most important biomarkers of prostate cancer. Because of their clinical importance, building PSA biosensing systems with high sensitivity and stability is essential. However, it still presents significant difficulties, such as low detection limits. We designed a platform to covalently bind the amino-terminated aptamer by modifying the ITO-PET surface with carboxyethylsilanetriol (CTES) to obtain a self-assembled monolayer (SAM). We also evaluated the potential for use in real human serum samples by investigating the optimal operating conditions and analytical performance characteristics of the developed biosensor. The design we present here exhibits excellent precision, with a limit of detection (LOD) as low as 8.74 fg/mL PSA. The broad linear detection range of the biosensor under optimal conditions was determined as 1.0-1500 fg/mL. The dissociation constant (Kd) for the aptamer was also calculated as 46.28 ± 5.63 nM by evaluating the impedimetric response as a function of PSA concentration. The aptasensor displayed considerable repeatability (1.3% RSD) and reproducibility (7.51% RSD) and good storage stability (98.34% of the initial activity for 8 weeks). Additionally, we demonstrated that the technique we developed was quite efficient in estimating the kinetics of aptamer-analyte interactions by determining the Kd and single-frequency impedance (SFI) data. In conclusion, we proposed a selective and sensitive biosensor with the potential for clinical application and superior performance in real serum samples.

MerR-fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water.

Mercury ion (Hg2+ ) is a universal pollutant and its detection is crucial for public healthcare. In this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury-sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+ -induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5-40 nM. The limit of detection was 0.5 nM, which was much lower than the maximum allowed level in water. The biosensor specificity was highly dependent on type and concentration of metal ion. The biosensor exhibited high specificity in a mixture of metal ions at 0.5 nM concentration. However, the interference effect was more pronounced at 40 nM concentration of metal ions. The measurement was completed in less than 1 Min with no need for sample preparation or preincubation steps. The biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry.

Development of genetically encoded fluorescent protein constructs of hyperthermophilic maltose-binding protein.

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.

A review of aptamer-conjugated nanomaterials for analytical sample preparation: Classification according to the utilized nanomaterials.

BACKGROUND: Sample extraction before detection is a critical step in analysis. Since targets of interest are often found in complex matrices, the sample can not be directly introduced to the analytical instrument. Nanomaterials with unique physical-chemical properties are excellent supports for use in sorbent-based extraction. However, they lack selectivity and thus need to be functionalized with target-capturing molecules. Antibodies and molecularly imprinted polymers (MIPs) can be used for this purpose, but they have some problems that limit their practical applications. Hence, functionalization of nanomaterials for selectivity remains a problem. RESULTS: Nucleic acid aptamers are affinity reagents that can provide superiority to antibodies since they can be selected in vitro and at a lower cost. Moreover, aptamers can be chemically synthesized and easily modified with different functional groups. Hence, aptamers are good candidates to impart selectivity to the nanomaterials. Recent studies focus on the integration of aptamers with magnetic nanoparticles, carbon-based nanomaterials, metal-organic frameworks, gold nanoparticles, gold nanorods, silica nanomaterials, and nanofibers. The unique properties of nanomaterials and aptamers make the aptamer-conjugated nanomaterials attractive for use in sample preparation. Aptamer-functionalized nanomaterials have been successfully used for selective extraction of proteins, small molecules, and cells from different types of complex samples such as serum, urine, and milk. In particular, magnetic nanoparticles have a wider use due to the rapid extraction of the sample under magnetic field. SIGNIFICANCE: In this review, we aim to emphasize how beneficial features of nanomaterials and aptamers could be combined for extraction or enrichment of the analytes from complex samples. We aim to highlight that the benefits are twofold in terms of selectivity and efficiency when employing nanomaterials and aptamers together as a single platform.

Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N-terminal of aspartate dehydrogenase.

We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima. The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni-chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate-induced conformational change was converted to a fluorescence signal by GFP. The recombinant protein responded to l-aspartate (l-Asp) linearly within the concentration range of 1-50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp in vitro, and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino-acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l-Asp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.

Lab-on-a-chip systems for cancer biomarker diagnosis.

Lab-on-a-chip (LOC) or micro total analysis system is one of the microfluidic technologies defined as the adaptation, miniaturization, integration, and automation of analytical laboratory procedures into a single instrument or "chip". In this article, we review developments over the past five years in the application of LOC biosensors for the detection of different types of cancer. Microfluidics encompasses chemistry and biotechnology skills and has revolutionized healthcare diagnosis. Superior to traditional cell culture or animal models, microfluidic technology has made it possible to reconstruct functional units of organs on chips to study human diseases such as cancer. LOCs have found numerous biomedical applications over the past five years, including integrated bioassays, cell analysis, metabolomics, drug discovery and delivery systems, tissue and organ physiology and disease modeling, and personalized medicine. This review provides an overview of the latest developments in microfluidic-based cancer research, with pros, cons, and prospects.

Fluid-based wearable sensors: a turning point in personalized healthcare.

Nowadays, it has become very popular to develop wearable devices that can monitor biomarkers to analyze the health status of the human body more comprehensively and accurately. Wearable sensors, specially designed for home care services, show great promise with their ease of use, especially during pandemic periods. Scientists have conducted many innovative studies on new wearable sensors that can noninvasively and simultaneously monitor biochemical indicators in body fluids for disease prediction, diagnosis, and management. Using noninvasive electrochemical sensors, biomarkers can be detected in tears, saliva, perspiration, and skin interstitial fluid (ISF). In this review, biofluids used for noninvasive wearable sensor detection under four main headings, saliva, sweat, tears, and ISF-based wearable sensors, were examined in detail. This report analyzes nearly 50 recent articles from 2017 to 2023. Based on current research, this review also discusses the evolution of wearable sensors, potential implementation challenges, and future prospects.

Biosensing strategies for diagnosis of prostate specific antigen.

Almost from the time of its discovery, the prostate specific antigen (PSA) has been one of the most accurate and most extensively studied indicators of prostate cancer (PC). Because of advancements in biosensing systems and technology, PSA analysis methods have been substantially updated and enhanced as compared to their first instances. With the development of techniques in biosensor technology, the number of PSA biosensors that can be used in the biomedical sector is increasing year by year. Many different recognition elements and transducers have been used in the development of biosensor systems that exhibit high sensitivity, selectivity, and specificity. Here in this review, we provide a current overview of the different approaches to PSA detection.

Single-stranded DNA (ssDNA) Aptamer targeting SipA protein inhibits Salmonella Enteritidis invasion of intestinal epithelial cells.

Salmonella Enteritidis is an important pathogen that can invade the intestinal cells of its host causing salmonellosis. SipA protein, an effector protein secreted by T3SS, maintains invasion of host cells more efficient. Thus, inhibitory aptamers against SipA protein were developed using magnetic bead-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. The enriched sequences were obtained after 9 SELEX rounds. Among which, an aptamer namely Apt17 displayed Kd values equivalent to 114.9 and 63.4 nM at 27 °C and 37 °C, respectively. The effect of Apt17 on adhesion and invasion of Caco-2 cells by the tested strains was determined. While the adhesion and invasion of Salmonella Enteritidis TM 6 were inhibited by 70% and 37.7%, those of Salmonella Enteritidis TM 68 were inhibited by 45.71% and 39.5% respectively. These results represent a corner stone for future studies that could aim to develop putative inhibitors against Salmonellosis.

A highly sensitive DNA aptamer-based fluorescence assay for sarcosine detection down to picomolar levels.

Sarcosine is an amino acid derivative, which is considered as a key metabolite in various metabolic processes. Therefore, simple and sensitive detection methods are needed for further understanding its metabolic role and diagnostic value. In this study, we developed a novel method that meets the need for practical and sensitive detection in a complex medium mimicking urine conditions. For this aim, we selected sarcosine-specific DNA aptamers using graphene oxide-assisted systemic evolution of ligands by exponential enrichment (GO-SELEX). The candidate aptamers were labeled with 6-carboxyfluorescein (6-FAM) at their 5' ends. Two aptamers, namely 9S and 13S produced a significant fluorescence signal upon sarcosine binding. Both aptamers enabled a sensitive analysis with a detection limit of 0.5 pM. The linear detection ranged between 5 pM and 50 µM for 9S aptamer, while 13S aptamer enabled a wider linear detection range between 5 pM and 500 µM. The aptamer-based assay allowed rapid detection with no need for chemical derivatization of sarcosine and sophisticated instruments. Moreover, the aptamer-based assay was free of interference from urea and human serum albumin.