Development of a DNA Microelectrochemical Biosensor for CEACAM5 Detection

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.

Transforming growth factor beta1 (TGF-beta1) is thought to be involved in cancer growth and progression. TGF-beta1 changes to its active form after being secreted in its latent form. Our aim was to clarify the significance of plasma concentrations of active and total TGF-beta1 of patients with colorectal cancer. Plasma concentrations of active and total TGF-beta1 in 45 patients with colorectal cancer and 23 healthy volunteers were measured using ELISA and the activation rate (ratio of active to total TGF-beta1) was determined. Plasma concentrations of active TGF-beta1 (21.9 +/- 12.8 pg/ml) were significantly higher in patients with colorectal cancer than in healthy volunteers (9.9 +/- 5.9 pg/ml; p < 0.001, Welch's t-test). Concentration of total TGF-beta1 was also significantly higher for patients with colorectal cancer (18.0 +/- 13.0 ng/ml vs. 11.1 +/- 6.4 ng/ml; p < 0.01, Welch's t-test). However, there was no significant difference in the TGF-beta1 activation rate between the 2 groups. There was a correlation between Dukes' stage and plasma concentration of active or total TGF-beta1 (p < 0.01, Spearman's rank correlation test) and on day 7 the active TGF-beta1 levels for patients recovering from curative resection were similar to those of the control group of healthy volunteers. These results suggest that active TGF-beta1 might be used as a tumor marker for colorectal cancer.

The goal of this study was to investigate Cystatin SN, a cysteine protease inhibitor, as a novel tumor marker for colorectal cancer (CRC). Gene expression profiles of mRNA from normal tissues and cancer cell lines were performed. Twenty-eight monoclonal antibodies for Cystatin SN were generated and serum Cystatin SN was quantified using ELISA in sera from 159 patients with CRC and 40 healthy controls. Cystatin SN was highly expressed in colon cancer cells. Employing a receiver-operating characteristic curve, we obtained an area under the curve of 0.708 for Cystatin SN, 0.819 for carcinoembryonic antigen (CEA) and 0.703 for carbohydrate antigen 19-9 (CA19-9). The combination assay of Cystatin SN, CEA and CA19-9 showed 62.9% sensitivity and 90.0% specificity. Especially, the sensitivity of the combination assay in stages I and II detection, in which stages curative operation would be possible, was improved over that of the assay testing only for CEA and CA19-9 (from 37.5 to 42.5% in stage I, from 49.0 to 60.8% in stage II). Furthermore, Western blot analysis revealed that Cystatin SN was increased in the urine from patients with CRC. Our results suggest the possibility of utilizing this novel tumor marker that can be tested in urine samples. These observations suggest that Cystatin SN in combination with CEA and CA19-9 is a useful tumor marker for detecting early stage CRC and that it is a unique urinary excretory protein, suggesting that Cystatin SN might be a novel candidate for use in mass screening for CRC.

Diclofenac, a widely used non-steroidal anti-inflammatory drug (NSAID), poses growing concerns in environmental contexts due to its potential toxicity and persistence. Accurate, real-time monitoring of diclofenac levels in both biological samples and water sources is essential to ensure therapeutic safety and reduce environmental impact. However, conventional methods such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) are limited by their complexity, high cost, and lack of portability for on-site testing.To address this need, we are developing a portable electrochemical biosensor capable of detecting diclofenac in both human biological fluids and environmental water samples. The biosensor is based on carbon screen-printed electrodes (SPEs) functionalized with didodecyldimethylammonium bromide (DDAB) and cytochrome P450 2C9 (CYP2C9) to enable selective and sensitive recognition of diclofenac. Electrochemical techniques—including cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS)—are employed to characterize sensor performance and detect drug–enzyme interactions. In parallel, UV absorbance spectroscopy at 340 nm was used to confirm the enzymatic activity of CYP2C9 through NADPH oxidation, ensuring the functional integrity of the immobilized enzyme.CV and EIS analyses confirmed successful biosensor fabrication and functional performance. Cyclic voltammetry showed increased current response with diclofenac concentration and clear electrochemical differences after DDAB and enzyme modification. DPV further demonstrated a concentration-dependent response, supporting the sensor’s analytical sensitivity. EIS revealed stepwise increases in charge transfer resistance, supporting effective electrode modification. Upon diclofenac exposure, a further increase in Rct was observed, indicating successful drug–enzyme interaction.By integrating enzyme-based selectivity with versatile electrochemical and spectroscopic techniques, this biosensor aims to provide a rapid, low-cost, and field-deployable solution for dual-purpose monitoring of diclofenac. This poster presents the rationale, sensor design, and analytical performance, emphasizing its potential to support both clinical diagnostics and environmental protection efforts.

In this paper, the implementation and characterization of a hand-held and simple biosensor for in-situ endotoxin determination are described. The integrated biosensor developed here is based on the electrochemical detection of endotoxin using polymyxin B as bioreceptor immobilized onto gold electrodes via a self-assembled monolayer. The cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy were used to characterize the biosensor performance and properties throughout the functionalization process. In addition, a comparative analysis of the behavior and features of two alternative electrochemical techniques for endotoxin detection was carried out. The biosensing device fabricated by thin-film technologies provided a simple and robust method to detect low concentrations of endotoxin.

In recent years, public awareness regarding the carbon dioxide emissions and the depletion of fossil fuel reserves has been increased. This motivates the development of the alternative energy sources based technology [1]. The Photoelectrochemical cell (PEC) is a capable clean and renewable way to convert sunlight into a storable chemical energy form. This technology enables the water to be split into hydrogen and oxygen by light-induced electrochemical processes [2]. One of the most studied and promising photocatalysts using in PEC is TiO2, which is relatively inexpensive and stable against photo irradiation while non-toxic [3]. In order to evaluate the PEC performances, the difference between various types of photocatalysts is usually attributed to the difference in their corresponding band gap energies, while the semiconductor particle size has not been taken into account as the main parameter. Having smaller particle size, particularly in the lower nanometer range, can give materials unique properties that could increase photoactivity [4]. There are some basic requirements for materials used as photo-electrodes such as optical function required to obtain maximal absorption of solar energy, the band gap of semiconductor which should be small enough to absorb a significant portion of the solar spectrum, the band edge potentials at the surfaces which must include the hydrogen and oxygen redox potentials, and especially sufficient charge transfer processes as well as the reaction kinetics at the semiconductor/electrolyte interface [5]. The present work reports the study of the kinetic aspects of photo electrochemical cells based on different nanoparticle sizes ranging from 50 nm down to 5 nm of TiO2, by using the electrochemical impedance spectroscopy (EIS) under illumination. The EIS is a useful method for the analysis of kinetic aspects such as charge transfer process that influences the cell performance. To measure the current response to the application of an AC voltage as a function of the frequency, EIS is considered as an electrochemical measurement [6]. Running the EIS is comparatively easy, but the interpretation of the results is often complicated and requires the application of suitable theoretical models. The equivalent electrical circuit is required to fit EIS data to identify the charge transfer phenomenon occurring in the PEC cell under 1 sun simulated illumination [5]. In our analysis, the three different electrode configurations were applied. The three semicircles are attributed to the redox reaction at the platinum counter electrode (Z1), the electron transfer at the TiO2 /electrolyte interface (Z2), and charge carrier transport in the electrolyte (Z3) (Figure 1a). The resistance elements R1, R2, and R3 are labeled as the real part of Z1, Z2, and Z3, respectively (Figure 1b). Therefore, it would be a powerful technique to recognize the internal resistance and the electron transportation of each photoelectrode in various particle size of the TiO2. A decrement of the internal resistance related to the electron transportation in the TiO2 /electrolyte interface causes an improvement in the electron transportation in the TiO2 thin film, which leads to the improvement of the cell efficiency. Furthermore, the exchange current density of each photoelectrode can be calculated when Rct (Charge Transfer Resistance) is known. This study is trying to reveal the effect of TiO2particle size on kinetic aspects of the Photoelectrochemical cell (PEC) for hydrogen production. Nanoparticle sizes ranging from 50 nm down to 5 nm were tested using EIS measurements. It was our prediction that by increasing particle size the internal resistance of electron transportation decreases, as per other literature [7]. The process of putting together the working electrode, as well as procedure and results of experiments, will be discussed.

Electron transfer studies on cholesterol LB films assembled on thiophenol and 2-naphthalenethiol self-assembled monolayers

A critical review of recent advancements in zinc based metal organic framework nanocomposites and their derivatives for supercapacitor applications with future perspectives and challenges

Rapid, simple, and sensitive analysis of relevant proteins is crucial in many research areas, such as clinical diagnosis and biomarker detection. In particular, clinical data on cancer biomarkers show great promise in forming reliable predictions for early cancer diagnostics, although the current analytical systems are difficult to implement in regions of limited recourses. Paper-based biosensors, in particular, have recently received great interest because they meet the criteria for point-of-care (PoC) devices; the main drawbacks with these devices are the low sensitivity and efficiency in performing quantitative measurements. In this work, we design a low-cost paper-based nanosensor through plasmonic calligraphy by directly drawing individual plasmonic lines on filter paper using a ballpoint pen filled with gold nanorods (AuNR) as the colloidal ink. The plasmonic arrays were further successively coated with negatively and positively charged polyelectrolyte layers employed as dielectric spacers to promote the enhancement of the emission of carboxyl-functionalized quantum dots (QD)—previously conjugated with specific antibodies—for indirect detection of the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5). The efficiency, sensitivity, as well as the specificity of our portable nanosensor were validated by recording the luminescence of the QD@Ab complex when different concentrations of CEACAM5 were added dropwise onto the calligraphed plasmonic arrays.

Self-assembled monolayers (SAMs) are widely used as surface modification structures for thin films synthesis and designed nanostructures fabrication1. Alkyltrichlorosilanes is a group of SAMs widely applied in the field of area-selective atomic layer deposition (AS-ALD). The closely packed octadecyltrichlorosilane (ODTS) SAMs could act as monolayer resist to prohibit ALD2, while the pinholes on unsaturated ODTS SAMs could act as nucleation sites to initiate ALD growth. Thus the distribution and structure of pinholes, including its size and density, are important factors for utilizing SAMs template to fabricate nanoparticles via ALD. Many studies characterize SAMs and pinholes by atomic force microscopy (AFM), scanning electron microscopy (SEM), scanning tunneling microscope (STM), etc3. Most of these methods could provide the microscopic information on the pinhole structures, yet the information from these methods is quite localized and hard to obtain statistical average and full-scale information about the pinhole size and distribution density. Here we report utilizing electrochemical impedance spectroscopy (EIS) to study the size distribution and loading of initial nanoparticles grown in those pinhole sites. EIS is a powerful and surface sensitive characterization technique to obtain the average and global characters of the detected surfaces4,5. In this study, the properties of unsaturated ODTS SAMs are characterized with EIS methods. The relationship between SAMs growth time and the pinholes properties has been established. With the SAMs immersing time increasing, the sizes and density distribution are both reduced. For SAMs sample immersed over long time, at least 24 hours, negligible pinholes are observed. Furthermore, Pd ALD process has been carried on those SAMs modified substrates. The distribution of Pd nanoparticles corresponds well with the pinholes. The results show that the nanoparticles grown on SAMs modified substrates depend on the pinholes properties. With SAMs modified substrate, ALD grown noble metal nanoparticles with uniform size and controlled loading density can be achieved.

Graphene-based electrochemical biosensors have emerged as promising tools for the early detection and monitoring of oral cancer through salivary biomarker analysis. Graphene's exceptional properties, including high surface area, superior electrical conductivity, and excellent mechanical strength, enable the development of highly sensitive and specific biosensors. This review provides a comprehensive overview of the current state-of-the-art in graphene-based electrochemical biosensors for salivary biomarker detection in oral cancer. We discuss the unique advantages of saliva as a diagnostic medium and highlight the key salivary biomarkers associated with oral cancer, including proteins, DNA, and RNA. Various electrochemical detection techniques, such as cyclic voltammetry, differential pulse voltammetry, electrochemical impedance spectroscopy, field-effect transistors, amperometry, chronoamperometry, and photoelectrochemical methods, are explored in the context of graphene-based biosensors. The challenges associated with the development and clinical translation of these biosensors are also addressed, emphasizing the need for improved functionalization strategies, enhanced stability, and standardized validation protocols. Finally, we present a futuristic outlook on the integration of graphene-based biosensors with artificial intelligence, microfluidics, and telemedicine platforms to enable personalized diagnostics and treatment monitoring. With continued advancements in sensor technology and computational tools, graphene-based electrochemical biosensors have the potential to revolutionize oral cancer management, improving patient outcomes and quality of life. • Offer sensitive detection of salivary biomarkers. • Saliva is a non-invasive diagnostic medium. • Employ various electrochemical techniques. • Challenges include complex cancer biology, low biomarker concentrations, and individual saliva composition variability. • Future advancements include integration with AI and microfluidics.

In this study, a measurement system based on Electrochemical Impedance Spectroscopy (EIS) was assembled to determine whether bacteriophage UP87 immobilized on a gold electrode surface would exhibit binding and lytic activity towards Aeromonas hydrophila. Bacteriophage UP87, a lytic phage for A. hydrophila, was immobilized on gold screen-printed electrodes (Au-SPEs), which contained a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) and dithiothreitol (DTT), via carbodiimide cross-linker chemistry. The phage solution incubation time on the Au SPE was optimized to ensure maximum phage coverage of the electrode. The optimum time for phage UP87 immobilization was 60 minutes. To confirm the successful immobilization of the phage on the electrode surface, the Au-SPEs were characterized using Fourier Transform Infrared Spectroscopy (FTIR). The study of the binding and lysis of A. hydrophila was done through bacterial solution incubation on the treated Au-SPEs for different durations. Binding of the bacterial cells was shown by the general increase in the charge transfer resistance (R ct ) up to a maximum value obtained after 60 minutes of incubation. Lysis of the captured bacterial cells was indicated through the decrease in R ct value after 80 minutes of incubation. In addition, the phage-modified Au-SPE exhibited higher R ct values at 60 minutes of incubation period for A. hydrophila compared with the non-target bacteria Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, thus showing satisfactory selectivity. This high selectivity was attributed to the pre-treatment of the Au-SPE which blocked nonspecific interactions and highly favored the binding of A. hydrophila. The results of this study indicate that surface immobilization of phages can be used to concentrate them for binding and lysing bacteria in solution. The concentration-dependence of the bacteria's binding to the phage-modified surface hints at the possibility of using the phage UP87 as a biorecognition element in a selective biosensor for A. hydrophila using the EIS technique.

According to our previously proposed scheme, each of three kinds of glycosphingolipid (GSL) derivatives, that is, lactosyl ceramide [Lac–Cer (1)] and gangliosides [GM1-Cer (2) and GT1b-Cer (3)], was installed onto the glass surface modified with Au nanoparticles. In the present study, we tried to apply microwave irradiation to promote their installing reactions. Otherwise, this procedure takes a lot of time as long as a conventional self-assembled monolayer (SAM) technique is applied. Using an advanced microwave reactor capable of adjusting ambient temperatures within a desired range, various GSL glycochips were prepared from the derivatives (1)–(3) under different microwave irradiation conditions. The overall assembling process was programed with an IC controller to finish in 1 h, and the derived GSL glycochips were evaluated in the analysis of three kinds of biological toxins [a Ricinus agglutinin (RCA120), botulinum toxin (BTX), and cholera toxin (CTX)] using a localized surface plasmon resonance (LSPR) biosensor. In the LSPR analysis, most of the irradiated GSL chips showed an enhanced response to the targeting toxin when they were irradiated under optimal temperature conditions. Lac–Cer chips showed the highest response to RCA120 (an agglutinin with β-D-Gal specificity) when the microwave irradiation was conducted at 30–35 °C. Compared to our former Lac–Cer glycochips with the conventional SAM condition, their response was enhanced by 3.6 times. Analogously, GT1b chips gained an approximately 4.1 times enhancement in their response to botulinum type C toxin (BTX/C) when the irradiation was conducted around at 45–60 °C. In the LSPR evaluation of the GM1-Cer glycochips using CTX, an optimal condition also appeared at around 30–35 °C. On the other hand, the microwave irradiation did not lead to a notable increase compared to the former GM1-Cer chips derived with the SAM technique. Judging from these experimental results, the microwave irradiation effectively promotes the installing process for all the three kinds of the GSL derivatives, while the optimal thermal condition becomes different from each other. Many bacterial and botanic proteinous toxins are composed of such carbohydrate binding domains or subunits that can discriminate both the key epitope structure and the dimension of glycoconjugates on the host cell surface. It is assumed that the optimal irradiation and thermal conditions are required to array these semi-synthetic GSL derivatives on the Au nanoparticles in a proper density and geometry for tight adhesion with each of the biological toxins.

A rapid surface modification technique for the formation of self-assembled monolayers (SAMs) of alkanethiols on gold thin films using microwave heating in <10 min is reported. In this regard, SAMs of two model alkanethiols, 11-mercaptoundecanoic acid (11-MUDA, to generate a hydrophilic surface) and undecanethiol (UDET, a hydrophobic surface), were successfully formed on gold thin films using selective microwave heating in (1) a semicontinuous fashion and (2) a continuous fashion at room temperature (24 h, control experiment, no microwave heating). The formation of SAMs of 11-MUDA and UDET was confirmed by contact angle measurements, Fourier transform infrared (FT-IR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The contact angles for water on SAMs formed by the selective microwave heating and conventional room temperature incubation technique (24 h) were measured to be similar for 11-MUDA and UDET. FT-IR spectroscopy results confirmed that the internal structures of SAMs prepared using both microwave heating and room temperature were similar. XPS results revealed that the organic and sulfate contaminants found on bare gold thin films were replaced by SAMs after the surface modification process had been conducted using both microwave heating and room temperature.

Two anion receptors, 1 and 2, based on the calix[6]crown-4 architecture were synthesized and characterized by NMR (1H, 13C, COSY), UV-vis, and MALDI-MS. 1H NMR measurements demonstrate that receptors 1 and 2 exhibit the highest binding affinity for fluoride ions compared to other anions including Cl-, Br-, NO3-, HSO4-, H2PO4-, and AcO-. The binding constants of 1 with F- and AcO- are 326 (+/-32) and 238 (+/-23) M-1, whereas those of 2 with F- and AcO- are 222 (+/-25) and 176 (+/-21) M-1. The fluorescent titration of 2 with various anions such as Cl-, Br-, NO3-, HSO4-, and H2PO4- led to essentially no change in excimer emission and a slight enhancement of monomer emission. In contrast, a dramatic change was observed in the fluorescence spectra upon the addition of F- and AcO- to 2. Self-assembled monolayers (SAMs) of 1 were formed on gold surfaces and characterized by reductive desorption and other techniques. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy were used to monitor anion recognition by the SAM-modified gold electrodes. The gold electrodes modified by SAMs of 1, upon binding with the F- anion, exhibit a dramatic increase in charge-transfer resistance (Rct) values. This is due to the repulsion between the negatively charged electrode surfaces and the negatively charged Fe(CN)6(3-/4-) redox probe in the electrolyte solution. In contrast, smaller increases in Rct values were observed in the cases of other monovalent anions investigated.